Treatment of CNS Disorders Associated with Mutations in Genes Encoding Lysosomal Enzymes

ABSTRACT

Described is a method for treating an individual having a neurological disorder with an associated mutation or mutations in a gene encoding a lysosomal enzyme. Specifically, the individual is administered a specific pharmacological chaperone for the lysosomal enzyme which increases trafficking of the protein from the ER to the lysosome in neural cells, with or without concomitantly increasing enzyme activity in neural cells. Restoration of trafficking relieves cell stress and other toxicities associated with accumulation of mutant proteins. Restoration of enzyme activity relieves substrate accumulation and pathologies associated with lipid accumulation. In a specific embodiment, the neurological disorder is Parkinson&#39;s disease or parkinsonism which is associated with mutations in glucocerebrosidase.

This application is a continuation of U.S. patent application Ser. No.14/506,118, filed Oct. 3, 2014, which is a divisional application ofU.S. patent application Ser. No. 12/986,506, filed Jan. 7, 2011, issuedas U.S. Pat. No. 8,883,813, which is a divisional application of U.S.patent application Ser. No. 11/449,528, filed Jun. 8, 2006, issued asU.S. Pat. No. 7,964,617, which claims priority from U.S. ProvisionalPatent Application Ser. No. 60/689,120, filed on Jun. 8, 2005, thedisclosures of each of which is incorporated herein by reference in itsentirety.

FIELD OF THE INVENTION

The present invention relates to a method for treating an individualhaving a neurological risk factor, condition, or disorder associatedwith a mutation or mutations in a lysosomal enzyme such as acidβ-glucosidase. Specifically, the individual is administered a specificpharmacological chaperone for the lysosomal enzyme which increasestrafficking of the protein from the ER to the lysosome in neural cells,and/or concomitantly increases enzyme activity in neural cells.

REFERENCE TO THE SEQUENCE LISTING

The Sequence Listing text file submitted herewith, identified as“00656772.TXT” (25 Kb, created Sep. 25, 2014), is hereby incorporated byreference.

BACKGROUND OF THE INVENTION

Lysosomal storage disorders are a group of autosomal recessive diseasescaused by the accumulation of cellular glycosphingolipids, glycogen, ormucopolysaccharides, due to defective hydrolytic enzymes. Examples ofLSDs include but are not limited to Gaucher disease (Beutler et al., TheMetabolic and Molecular Bases of Inherited Disease, 8th ed. 2001 Scriveret al., ed. pp. 3635-3668, McGraw-Hill, New York), G_(m1)-gangliosidosis(id. at pp 3775-3810), fucosidosis (The Metabolic and Molecular Bases ofInherited Disease 1995. Scriver, C. R., Beaudet, A. L., Sly, W. S. andValle, D., ed pp. 2529-2561, McGraw-Hill, New York),mucopolysaccharidoses (id. at pp 3421-3452), Pompe disease (id at pp.3389-3420), Hurler-Scheie disease (Weismann et al., Science. 1970; 169,72-74), Niemann-Pick A and B diseases, (The Metabolic and MolecularBases of Inherited Disease 8th ed. 2001. Scriver et al. ed., pp3589-3610, McGraw-Hill, New York), and Fabry disease (id at pp.3733-3774). Others include Metachromatic Leukodystrophy, Kuf's Disease(Adult Neuronal Lipoid Lipofucsinosis) and Adrenoleukodystrophy. EachLSD is associated with a specific defective hydrolytic enzyme caused byone or more mutations which cause the enzyme to become conformationallyunstable in the ER following synthesis, and thus, become targeted fordegradation instead of trafficking through the Golgi to the nativelocation in the lysosome.

Several LSDs have significant neurological involvement. For example,Gaucher disease is the most common LSD that is associated with theaccumulation of glycosphingolipids (GSL) in cells, particularlymonocytes and macrophages, of afflicted individuals. This aberrant buildup of GSL results from a genetic deficiency (mutation) in the lysosomalenzyme acid β-glucosidase (Gba; glucocerebrosidase), the lysosomalhydrolase that breaks down the GSL glucosylceramide (GluCer). Thedisease has been classified into three clinical types, depending onneurological involvement and disease severity (Cox et al., Q J Med.2001; 94: 399-402). Type 2 Gaucher disease is the rarest, most severeform, and is associated with early onset of acute neurologic disease.The characteristic feature of neuronopathic Gaucher disease is anabnormality of horizontal gaze. Afflicted patients develop progressiveencephalopathy and extrapyrimidal symptoms such as rigidity andParkinson's-like movement (parkinsonism). Most Type 2 Gaucher patientsdie in early childhood from apnea or aspiration due to neurologicaldeterioration.

Type 3 Gaucher disease also has neurological involvement, although to alesser extent than Type 2. Type 3 patients have central nervous systemsymptoms that include poor coordination of movements (ataxia), seizures,paralysis of the eye muscles, epilepsy, and dementia. Asub-classification of Type 3, Type 3c, is associated withhepatosplenomegaly, corneal opacities, progressive ataxia and dementia,and cardiac valve and aortic root calcification.

Other LSDs with neurological involvementinclude G_(M1) gangliosidosis,which is associated with mutant β-galactosidase and results in neuronallipidosis; G_(M2) gangliosidosis (Tay-Sachs disease), which isassociated with mutant hexosaminidase A and results in neuronallipidosis; Niemann-Pick Disease, which is associated with mutantsphingomyelinase and also results in neuronal lipidosis; (Krabbedisease) galactocerebrosidase leukodystrophy; and neuronal ceroidlipofuscinoses, which is associated with mutant lysosomal proteases andresults in neuronal lipidosis. Metachromatic Leukodystrophy is adeficiency of the enzyme arylsulfatase A and patients' symptoms includeprogressive movement disorders, seizures, cognitive disorders and alsoschizophrenia and psychiatric problems in addition to gastrointestinaldisturbances. Kuf's Disease (Adult Neuronal Lipoid Lipofucsinosis) canmanifest as psychiatric symptoms and seizures. Adrenal Leukodystrophy isa disorder which is characterized by progressive white-matterdemyelination of the central nervous system and adrenocorticalinsufficiency.

Specific Pharmacological Chaperones

Recently, a specific pharmacological chaperone strategy has beendeveloped to rescue unstable, mutated proteins from degradationpresumably in the endoplasmic reticulum (ER) or in other cellularprotein degradation/disposal systems. In particular embodiments, thisparadigm shifting strategy employs small molecule reversible inhibitorswhich specifically bind to a defective lysosomal enzyme associated witha particular lysosomal disorder, stabilize the mutant enzyme in the ER,and “chaperone” the mutant enzyme so that it exits the ER. It wasunexpectedly found that the inhibitors could bind with specificity tothe enzyme during synthesis and folding in the ER, but could dissociatefrom the enzyme at its native location, thereby restoring its activity.In the absence of the chaperone, the mutated enzyme protein foldsimproperly in the ER (Ishii et al., Biochem. Biophys, Res. Comm. 1996;220: 812-815), is retarded in its maturation to a final product, and issubsequently degraded in the ER. These specific chaperones aredesignated specific pharmacological chaperones (or active site-specificchaperones where the chaperone is a competitive inhibitor of an enzyme).

The term “active site-specific chaperone” evolved from initial studiesusing wild-type and mutant lysosomal enzymes. The catalytic portion ofenzymes, i.e., the part where the enzyme binds to and interacts with itssubstrate, is generally known as the “active site in.” Thecounterintuitive strategy of using a reversible competitive inhibitor ofan enzyme (i.e., an enzyme inhibitor which competes with the substratefor binding to the catalytic center) to induce misfolded lysosomalenzymes to assume a stable molecular conformation, was firsthypothesized by virtue of the ability of some competitive inhibitors tobind the catalytic centers during biosynthesis and stabilize enzymes.Thus, any stabilization that could be achieved in vivo in the ER duringfolding of a nascent enzyme, especially a mutant enzyme having a foldingdefect, would be beneficial since it would prevent binding of theendogenous ER “chaperones” that bind misfolded polypeptides and targetthem for degradation. Moreover, the competitive inhibitor was“reversible” as it dissociated from the enzyme once the enzyme reachedthe lysosome, where the inhibitor was out-competed by natural substrate.

The specific chaperone strategy has been described and exemplified forabout 15 enzymes involved in LSDs in U.S. Pat. Nos. 6,274,597,6,583,158, 6,589,964, and 6,599,919, to Fan et al., which areincorporated herein by reference in their entirety. For example, a smallmolecule derivative of galactose, 1-deoxygalactonojirimycin (DGJ), apotent competitive inhibitor of the mutant Fabry enzyme α-galactosidaseA (α-Gal A), effectively increased in vitro stability of the humanmutant α-Gal A (R301Q) at neutral pH, and it enhanced the mutant enzymeactivity in lymphoblasts established from Fabry patients with R301Q orQ279E mutations. Furthermore, oral administration of DGJ to transgenicmice overexpressing a mutant (R301Q) α-Gal A substantially elevated theenzyme activity in major organs (Fan et al., Nature Med. 1999; 5:112-115). Similar rescue of Gba from Gaucher patient cells has beendescribed using another iminosugar, isofagomine (IFG), and itsderivatives, described in U.S. Pat. No. 6,916,829 to Fan et al., andusing other compounds specific for Gba (described in pending U.S. patentapplication Ser. Nos. 10/988,428, and 10/988,427, both filed Nov. 12,2004).

LSD Enzyme Mutations and Neurological Disorders

Gba and Parkinson's. It has recently been discovered that there is alink between mutations in lysosomal enzymes and neurological disordersother than the LSDs. As one example, there is a well-established linkbetween mutations in the Gba gene and Parkinson's disease. In one study,a group of 17 patients with rare, early onset, treatment-resistantparkinsonism were found to have at least one allele with a Gba missensemutation, including homozygous and heterozygous individuals for N370S, amutation typically associated with type 1, non-neuronopathic disease(Tayebi et al., Mol. Genet. Metab. 2003; 79; 104-109). In another study,a population of 99 Ashkenazi Jews with idiopathic Parkinson's diseasewere evaluated for six Gba mutations (N370S, L444P, 84GG, V394L, andR496H). Thirty-one Parkinson's patients had one or two mutant Gbaalleles: 23 were heterozygous for N370S; 3 were homozygous for N370S; 4were heterozygous for 84 GG; and 1 was heterozygous for R496H(Aharon-Peretz et al., New Eng. J Med 2004; 351: 1972-77). The frequencyof a mutant N370S allele was 5 times that among 1573 normal subjects,and that of 84GG was 21 times that of normal subjects. Among patientswith Parkinson's disease, patients carrying a Gba mutation also wereyounger than those who were not carriers. This study suggests thatheterozygosity for a Gba mutation may predispose Ashkenazi Jews toParkinson's disease.

Parkinson's and Gaucher diseases also share some pathological features,including neuronal loss, astrogliosis, and the presence of cytotoxicLewy-body-like α-synuclein inclusions in hippocampal neurons (the CA2-4region). A recent publication described the extent of neurologicalpathology in all three forms of Gaucher disease (Wong et al., Mol.Genet. Metabol. 2004; 38: 192-207). Abnormalities in cerebral corticallayers 3 and 5, hippocampal CA2-4, and layer 4b were found in Gaucherpatients having all three types. Neuronal loss was evident only inpatients with types 2 and 3, whereas type 1 patients presented withastrogliosis (Wong et al., supra). Two patients with type 1 Gaucher andparkinsonism/dementia exhibited α-synuclein positive inclusions inhippocampal CA2-4 neurons, one patient had brainstem-type andcortical-type Lewy bodies, and one had marked neuronal loss ofsubstantia nigra neurons (Wong et al., supra). In summary, all 4patients with parkinsonism and dementia had hippocampal CA2-4 gliosis,and neuronal depletion, gliosis, and brainstem-type Lewy bodies in thesubstantia nigra.

Several mouse models also demonstrate this link between Gba andParkinson's. The optimal in vitro hydrolase activity of Gba requiressaposin C, an activator protein that derives from a precursor,prosaposin. Transgenic mice expressing low levels (4-45% of wild type)of prosaposin and saposins (PS-NA), backcrossed into mice with specificpoint mutations (V394L/V394L or D409H/D409H) of Gba, has several CNSphenotypes similar to PD phenotypes including: gait ataxia, tremor,shaking to the point of falling over, and a neurogenic bladder (Sun etal., J Lipid Res. 2005. 46(10): 2102-13).

The specific pharmacological chaperone work described above establishedthe ability to restore enough function to a mutant enzyme(conformational mutation) to reduce or even eliminate the build-up oftoxic quantities of lipid substrate in the LSDs. However, it was notclear that this approach could affect heterozygous individuals, orindividuals with homozygous mutations who are not diagnosed with an LSDaccording to current criteria, but are at risk of developing aneurological condition or disorder due to the effects of the mutation,or individuals who are diagnosed with having lysosomal storage disordersbut have mutations in addition to or other than conformational mutationswhich render the protein non-functional. All of these populations are atrisk of developing a neurological disorder due to either toxic gain offunction, pathologic loss of function, or a combination. Thus, thereremains a need in the art to be able to identify causative factors andaddress the consequences of such mutations in these patient populations.

SUMMARY OF THE INVENTION

The present invention provides a method for the treatment of aneurological disorder in an individual, wherein the neurologicaldisorder is associated with a mutation in the gene encoding a lysosomalenzyme, by administering an effective amount of a specificpharmacological chaperone to treat the neurological disorder.

In one embodiment, the individual is homozygous for the mutation. Inanother embodiment, the individual is hemizygous, heterozygous orcompound heterozygous for the mutation.

In one embodiment, the mutation results in the enzyme being aconformational mutant.

In a specific embodiment, wherein the chaperone increases trafficking ofthe mutant enzyme from the endoplasmic reticulum and may or may notconcomitantly restore enzyme activity.

In another embodiment, the mutation results in increased amounts of, oraggregation, of another cellular substance, such as a lipid or anotherprotein or protein fragment, such as α-synuclein.

In a specific embodiment of the present invention, the lysosomal enzymeis glucocerebrosidase and the neurological disorder is Parkinson'sdisease or parkinsonism.

In another specific embodiment, the Parkinson's disease is early-onsetParkinson's disease.

In some embodiments of the invention, the specific pharmacologicalchaperone is an inhibitor of the lysosomal enzyme, and the inhibitor isa reversible or competitive inhibitor or both.

In a specific embodiment, the pharmacological chaperone forglucocerebrosidase is isofagomine or (5R, 6R, 7S,8S)-5-hydroxymethyl-2-octyl-5,6,7,8-tetrahydroimidazo[1,2-a]pyridine-6,7,8-triol.

The present invention also provides a method for diagnosing aneurological disorder associated with a mutant lysosomal enzyme, byscreening an individual who exhibits neurological symptoms for amutation in one or more lysosomal enzymes.

In one embodiment, the mutation results in an enzyme that is aconformational mutant.

In another embodiment, the screening is done by determining decreasedenzyme activity from a biological sample from the individual comparedwith a biological sample from a healthy individual.

In a specific embodiment, the neurological disorder diagnosed isparkinsonism or Parkinson's disease.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-B. FIG. 1 demonstrates the levels of Gba activity in the brainsfrom L444P transgenic mice treated with the specific pharmacologicalchaperone isofagomine (1A). Also depicted is the Gba activity level inthe brain following a washout and retreatment period (1B).

FIGS. 2A-N. FIG. 2 depicts fluorescent staining of lysosomes usingLysoTracker® Red in cells from Gaucher fibroblasts (2A) and normalfibroblasts (2B). Staining for lysosomal protein LAMP-1 was alsoperformed on normal fibroblasts (2C) and Gaucher fibroblasts (2D). FIG.2E-F shows an overlay of dual Gba and LAMP-1 staining in Gaucherfibroblasts. Also depicted is a dual overlay (LAMP-1 and Gba) of Gauchercells treated with the specific pharmacological chaperone isofagomine(2G-H) and the specific pharmacological chaperone C-benzyl-isofagomine(2I-J). Lastly, FIGS. 2K-N show staining of Gaucher cells for Gba only.Control Gaucher cells were stained with secondary antibody only (2K), orwere not treated (2L), or were treated with isofagomine (2M) orC-benzyl-isofagomine (2N).

FIGS. 3A-I. FIG. 3 depicts fluorescent staining of Gaucher cells (3D-I)and normal fibroblasts (3A-C) for the presence of polyubiquinatedproteins (PUP) (3A, 3D, 3G) and Gba (3B, 3E, 3H), and an overlay forboth (3C, 3F, 3I).

FIG. 4. FIG. 4 depicts the gene encoding human acid β-glucosidase, alsoreferred to as glucocerebrosidase or Gba (GenBank Accession No. J03059;SEQ ID NO: 1).

FIG. 5. FIG. 5 depicts the wild-type human Gba protein. The Gba proteinconsists of 536 amino acids and is in GenBank Accession No. J03059 (SEQID NO: 2).

FIG. 6. FIG. 6 depicts the homologous pseudogene for Gba located about16 kb downstream of the Gba gene (GenBank Accession No. M16328; SEQ IDNO: 3).

FIG. 7, FIG. 7 depicts the polypeptide encoded by the homologouspseudogene for Gba (SEQ ID NO: 4).

DETAILED DESCRIPTION

The present invention is based on the discovery that neurologicaldisorders presenting in individuals not diagnosed with lysosomal storagedisorders may be linked to mutations in lysosomal enzymes. Accordingly,the present invention, a specific pharmacologic chaperone, such as anASSC, can ameliorate both gain of function and loss of functionpathologies associated with mutations of lysosomal enzymes which arelinked with neurological risk factors, conditions, or disorders. Thechaperones can induce proper trafficking of mutant proteins at asufficient level to inhibit, even to the point of prevention, toxicaccumulation associated with the build up of misfolded, mutant proteins(i.e., gain of function), which in turn can effect neurologicalfunction. In some cases where the mutation only impairs folding andtrafficking of the protein to its native cellular location and is not,e.g., a mutant which impairs catalytic or other activity of the protein,or is a nonsense mutant, the chaperones also can restore activity to themutant protein, thereby addressing pathologies associated with theprotein's loss of function, such as substrate accumulation or evenaggregation of other toxic proteins or fragments which results fromaccumulation of substrate.

Definitions

The terms used in this specification generally have their ordinarymeanings in the art, within the context of this invention and in thespecific context where each term is used. Certain terms are discussedbelow, or elsewhere in the specification, to provide additional guidanceto the practitioner in describing the compositions and methods of theinvention and how to make and use them.

The term “Gaucher disease” includes Type 1, Type 2 and Type 3, andintermediates and subgroups thereof based on phenotypic manifestations.

A “neurological disorder” refers to any central nervous system (CNS) orperipheral nervous system (PNS) disease that is associated with neuronalor glial cell defects including but not limited to neuronal loss,neuronal degeneration, neuronal demyelination, gliosis (i.e.,astrogliosis), or neuronal or extraneuronal accumulation of aberrantproteins or toxins (e.g., β-amyloid, or α-synuclein). The neurologicaldisorder can be chronic or acute. Exemplary neurological disordersinclude but are not limited to Gaucher disease and other LSDs includingFabry disease, Tay-Sachs disease, Pompe disease, and themucopolysaccharidoses; Parkinson's disease; Alzheimer's disease;Amyotrophic Lateral Sclerosis (ALS); Multiple Sclerosis (MS);Huntington's disease; Fredrich's ataxia; Mild Cognitive Impairment; andmovement disorders (including ataxia, cerebral palsy, choreoathetosis,dystonia, Tourette's syndrome, kernicterus); tremor disorders,leukodystrophies (including adrenoleukodystrophy, metachromaticleukodystrophy, Canavan disease, Alexander disease, Pelizaeus-Merzbacherdisease); neuronal ceroid lipofucsinoses; ataxia telangectasia; and RettSyndrome. This term also includes cerebrovascular events such as strokeand ischemic attacks.

As used herein, the term “neurological disorder” also includes personsat risk of developing a neurological disorder, disease or condition aswell as persons already diagnosed with a neurological disorder, diseaseor condition.

A “neurological disorder associated with a mutation in a lysosomalenzyme” refers to any neurological disorder in which a mutation ormutations in the gene encoding the enzyme are also present when assessedin individuals having the neurological disorder, compared withindividuals not having or at risk of developing the neurologicaldisorder (i.e., healthy individuals). In specific embodiments, theneurological disorder associated with Gba (Gaucher) mutations isParkinson's disease or parkinsonism.

The term “human Gba gene” refers to the gene encoding acidβ-glucosidase, also referred to as glucocerebrosidase or Gba, The Gbagene is on chromosome 1q21 and involves 11 exons (GenBank Accession No.J03059; SEQ ID NO: 1). There is also a homologous pseudogene for Gbalocated about 16 kb downstream of the Gba gene (GenBank Accession No.M16328; SEQ 1D NO: 3).

The “human Gba” protein refers to the wild-type human Gba protein. TheGba protein consists of 536 amino acids and is in GenBank Accession No.J03059 (SEQ ID NO: 2). The polypeptide encoded by the above-referencedpseudogene is depicted in SEQ ID NO: 4.

As used herein, the term “pharmacological chaperone,” or sometimes“specific pharmacological chaperone” (“SPC”), refers to a molecule thatspecifically binds to a protein such as a lysosomal enzyme (e.g., Gba)and has one or more of the following effects: (i) enhancing theformation of a stable molecular conformation of the protein; (ii)inducing trafficking of the protein from the ER to another cellularlocation, preferably a native cellular location, i.e., preventingER-associated degradation of the protein; (iii) preventing aggregationof misfolded proteins; (iv) restoring or enhancing at least partialwild-type function, stability, and/or activity to the protein; and/orimproving the phenotype or function of the cell harboring the protein.Thus, a pharmacological chaperone for a protein is a molecule that bindsto the protein resulting in proper folding, trafficking,non-aggregation, and activity of the protein. As used herein, this termdoes not refer to endogenous chaperones, such as BiP, or to non-specificagents which have demonstrated non-specific chaperone activity againstvarious proteins, such as glycerol, DMSO or deuterated water, sometimescalled “chemical chaperones” (see Sato et al., Biochem Biophys Acta.1988; 126(2): 756-62; Welch et al., Cell Stress and Chaperones 1996;1(2):109-115; Welch et al., Journal of Bioenergetics and Biomembranes1997; 29(5):491-502; U.S. Pat. No. 5,900,360; U.S. Pat. No. 6,270,954;and U.S. Pat. No. 6,541,195).

As used herein, the term “specifically binds” refers to the interactionof a pharmacological chaperone with a specific protein, specifically, aninteraction with amino acid residues of a protein that directlyparticipate in contacting the pharmacological chaperone. A compound thatspecifically binds to a target protein, e.g., Gba, means that it bindsto and exerts a pharmacological chaperone effect on Gba and not ageneric group of related or unrelated proteins. The amino acid residuesof the protein that interact with any given pharmacological chaperonemay or may not be within the protein's “active site.” Specific bindingcan be evaluated through routine binding assays or through structuralstudies, e.g., co-crystallization, NMR, and the like.

The term “wild-type protein” refers to the nucleotide sequences encodingfunctional proteins, and polypeptide sequences encoded by theaforementioned nucleotide sequences, and any other nucleotide sequencesthat encode a functional polypeptide (having the same functionalproperties and binding affinities as the aforementioned polypeptidesequences), such as allelic variants in normal individuals, that havethe ability to achieve a functional conformation in the ER, achieveproper localization within the cell, and exhibit wild-type activity(e.g., GluCer hydrolysis).

As used herein the term “mutant protein” refers to a polypeptidetranslated from a gene containing a genetic mutation that results in analtered amino acid sequence. In one embodiment, the mutation results ina protein that does not achieve a native conformation under theconditions normally present in the ER, when compared with wild-typeprotein, or exhibits decreased stability or activity as compared withwild-type protein. This type of mutation is referred to herein as a“conformational mutation,” and the protein bearing such a mutation isreferred as a “conformational mutant.” The failure to achieve thisconformation results in the protein being degraded or aggregated, ratherthan being transported through a normal pathway in the protein transportsystem to its native location in the cell or into the extracellularenvironment.

In another embodiment, the protein has another mutation in addition toor other than a conformational mutant, which permits translation, andhence ER accumulation of all or portion of the protein (which proteinmay or may not retain wild-type activity).

In some embodiments, a mutation may occur in a non-coding part of thegene encoding the protein that results in less efficient expression ofthe protein, e.g., a mutation that affects transcription efficiency,splicing efficiency, mRNA stability, and the like. By enhancing thelevel of expression of wild-type as well as conformational mutantvariants of the protein, administration of a pharmacological chaperonecan ameliorate a deficit resulting from such inefficient proteinexpression.

Other mutations can result in decreased enzymatic activity or a morerapid turnover.

Specific embodiments of Gba mutants associated with neuronopathicdiseases include but are not limited to: N370S, L444P, K198T, D409H,R496H, V394L, 84GG, and R329C.

A heterozygous mutation of Gba refers to a genotype in which there isone wild-type allele and one mutant allele, e.g., N370S/wt. Aheterozygous Gba mutation also refers to a genotype in which there aretwo mutated alleles, each with a different mutation, e.g., N370S/L444P.This term also includes the mutant/null genotype, i.e., N370S/null. Thisdefinition is also applicable when referring to heterozygous mutationsin other lysosomal enzymes.

A homozygous Gba mutation refers to a genotype in which there are twomutant Gba alleles in which the mutations are same, e.g., N370S/N370S.This definition is also applicable when referring to homozygousmutations in other lysosomal enzymes.

The term “stabilize a proper conformation” refers to the ability of apharmacological chaperone to induce or stabilize a conformation of amutated protein that is functionally identical to the conformation ofthe wild-type counterpart. The term “functionally identical” means thatwhile there may be minor variations in the conformation (almost allproteins exhibit some conformational flexibility in their physiologicalstate), conformational flexibility does not result in (1) proteinaggregation, (2) elimination through the endoplasmicreticulum-associated degradation pathway, (3) impairment of proteinfunction, e.g., Gba activity, and/or (4) improper transport within thecell, e.g, localization to the lysosome, to a greater or lesser degreethan that of the wild-type protein.

The term “stable molecular conformation” refers to a conformation of aprotein, i.e., Gba, induced by a specific pharmacological chaperone,that provides at least partial wild-type function in the cell. Forexample, a stable molecular conformation of a mutant protein would beone where the protein escapes from the ER and trafficks to the nativecellular location as does a wild-type Gba (e.g., the lysosome), insteadof misfolding and being degraded. In addition, a stable molecularconformation of a mutated protein may also possess full or partialactivity, e.g., GluCer hydrolysis. However, it is not necessary that thestable molecular conformation have all of the functional attributes ofthe wild-type protein.

The term “wild-type activity” refers to the normal physiologicalfunction of a protein, e.g., Gba, in a cell. For example, Gba activityincludes folding and trafficking from the ER to the lysosome, with orwithout the concomitant ability to hydrolyze a substrate such as GluCeror 4-methylumbelliferyl (4-MU). Such fimctionality can be tested by anymeans known to establish functionality of such a protein.

Certain tests may evaluate attributes of a protein that may or may notcorrespond to its actual in vivo function, but nevertheless areaggregate surrogates of protein functionality, and wild-type behavior insuch tests is an acceptable consequence of the protein folding rescue orenhancement _(t)echniques of the invention. One such activity inaccordance with the invention is appropriate transport of a mutantprotein, e.g., Gba from the endoplasmic reticulum to the native cellularlocation e.g., lysosome, or into the extracelluar environment.

The term “endogenous expression” refers to the normal physiologicalexpression of a protein in cells in an individual not having orsuspected of having a CNS disease or disorder associated with adeficiency, overexpression, or other defect, of a protein such as in thenucleic acid or polypeptide sequence which inhibit its expression,activity, or stability. This term also refers to the expression of theprotein in cell types in which it is normal for the protein to beexpressed and does not include expression in cells or cell types, e.g.,tumors, in which the protein is not expressed in healthy individuals.

As used herein, the terms “enhance expression” or “increase expression”refer to increasing the amount of a polypeptide that adopts a functionalconformation in a cell contacted with a pharmacological chaperonespecific for that protein, relative to its expression in a cell(preferably of the same cell-type or the same cell, e.g., at an earliertime) not contacted with the pharmacological chaperone specific for thatprotein. The aforementioned terms alternatively mean increasing theefficiency of transport of a polypeptide from the ER in a cell contactedwith a pharmacological chaperone specific for that protein, relative tothe efficiency of transport of a wild-type counterpart in a cell(preferably of the same cell, e.g., at an earlier time, or the same celltype) not contacted with the pharmacological chaperone specific for thatprotein.

As used herein, the term “efficiency of transport” refers to the abilityof a mutant protein to be transported out of the endoplasmic reticulumto its native location within the cell, to another location within thecell, to the cell membrane, or into the extracellular environment.

A “competitive inhibitor” of an enzyme can refer to a compound whichstructurally resembles the chemical structure and molecular geometry ofthe enzyme substrate to bind the enzyme in approximately the samelocation as the substrate, Thus, the inhibitor competes for the sameactive site as the substrate molecule, thus increasing the Km.Competitive inhibition is usually reversible if sufficient substratemolecules are available to displace the inhibitor, i.e., competitiveinhibitors can bind reversibly. Therefore, the amount of enzymeinhibition depends upon the inhibitor concentration, substrateconcentration, and the relative affinities of the inhibitor andsubstrate for the active site.

Non-classical competitive inhibition occurs when the inhibitor bindsremotely to the active site, creating a conformational change in theenzyme such that the substrate can no longer bind to it. Innon-classical competitive inhibition, the binding of substrate at theactive site prevents the binding of inhibitor at a separate site andvice versa. This includes allosteric inhibition.

A “linear mixed-type inhibitor” of an enzyme is a type of competitiveinhibitor that allows the substrate to bind, but reduces its affinity,so the Km is increased and the Vmax is decreased.

A “non-competitive inhibitor” refers to a compound that forms strongbonds with an enzyme and may not be displaced by the addition of excesssubstrate, i.e., non-competitive inhibitors may be irreversible. Anon-competitive inhibitor may bind at, near, or remote from the activesite of an enzyme or protein, and in connection with enzymes, has noeffect on the Km but decreases the Vmax. Uncompetitive inhibition refersto a situation in which inhibitor binds only to the enzyme-substrate(ES) complex. The enzyme becomes inactive when inhibitor binds. Thisdiffers from non-classical competitive inhibitors which can bind to theenzyme in the absence of substrate.

The term “Vmax” refers to the maximum initial velocity of an enzymecatalyzed reaction, i.e., at saturating substrate levels. The term “Km”is the substrate concentration required to achieve ½ Vmax.

A “responder” is an individual diagnosed with a neurological disorderassociated with a lysosomal enzyme mutation and treated according to thepresently claimed method who exhibits an improvement in, amelioration,or prevention of, one or more clinical symptoms, or improvement orreversal of one or more surrogate clinical markers. As one example, a“responder” for individuals with Parkinson's disease (having concomitantGba mutations) is one who exhibits improvement in, amelioration, orprevention of, one or more clinical symptoms, or improvement or reversalof one or more surrogate clinical markers including but not limited to:neuronal loss, astrogliosis, and the presence of intraneuronalLewy-body-like a-synuclein inclusions in CA2-3 neurons.

The terms “therapeutically effective dose” and “effective amount” referto the amount of the specific pharmacological chaperone that issufficient to result in a therapeutic response. A therapeutic responsemay be any response that a user (e.g., a clinician) will recognize as aneffective response to the therapy, such as by assessing symptoms andsurrogate clinical markers. Thus, a therapeutic response will generallybe an amelioration of one or more symptoms of a disease or disorder,e.g., a neurological disorder.

The phrase “pharmaceutically acceptable” refers to molecular entitiesand compositions that are physiologically tolerable and do not typicallyproduce untoward reactions when administered to a human. Preferably, asused herein, the term “pharmaceutically acceptable” means approved by aregulatory agency of the federal or a state government or listed in theU.S. Pharmacopeia or other generally recognized pharmacopeia for use inanimals, and more particularly in humans. The term “carrier” refers to adiluent, adjuvant, excipient, or vehicle with which the compound isadministered. Such pharmaceutical carriers can be sterile liquids, suchas water and oils. Water or aqueous solution saline solutions andaqueous dextrose and glycerol solutions are preferably employed ascarriers, particularly for injectable solutions. Suitable pharmaceuticalcarriers are described in “Remington's Pharmaceutical Sciences” by E. W.Martin, 18th Edition, or other editions.

The terms “about” and “approximately” shall generally mean an acceptabledegree of error for the quantity measured given the nature or precisionof the measurements. Typical, exemplary degrees of error are within 20percent (%), preferably within 10%, and more preferably within 5% of agiven value or range of values. Alternatively, and particularly inbiological systems, the terms “about” and “approximately” may meanvalues that are within an order of magnitude, preferably within 5-foldand more preferably within 2-fold of a given value. Numerical quantitiesgiven herein are approximate unless stated otherwise, meaning that theterm “about” or “approximately” can be inferred when not expresslystated.

Molecular Biology Definitions

In accordance with the present invention there may be employedconventional molecular biology, microbiology, and recombinant DNAtechniques within the skill of the art. Such techniques are explainedfully in the literature. See, e.g., Sambrook, Fritsch & Maniatis,Molecular Cloning: A Laboratory Manual, Second Edition (1989) ColdSpring Harbor Laboratory Press, Cold Spring Harbor, New York (herein“Sambrook et al., 1989”); DNA Cloning: A Practical Approach, Volumes Iand II (D. N. Glover ed. 1985); Oligonucleotide Synthesis (M. J. Gaited. 1984); Nucleic Acid Hybridization [B. D. Hames & S. J. Higgins eds.(1985)]; Transcription And Translation [B. D. Hames & S. J. Higgins,eds. (1984)]; Animal Cell Culture [R. I. Freshney, ed. (1986)];Immobilized Cells And Enzymes [IRL Press, (1986)]; B. Perbal, APractical Guide To Molecular Cloning (1984); F. M. Ausubel et al.(eds.), Current Protocols in Molecular Biology, John Wiley & Sons, Inc.(1994).

As used herein, the term “isolated” means that the referenced materialis removed from the environment in which it is normally found. Thus, anisolated biological material can be free of cellular components, i.e.,components of the cells in which the material is found or produced. Inthe case of nucleic acid molecules, an isolated nucleic acid includes aPCR product, an isolated mRNA, a cDNA, or a restriction fragment. Inanother embodiment, an isolated nucleic acid is preferably excised fromthe chromosome in which it may be found, and more preferably is nolonger joined to non-regulatory, non-coding regions, or to other genes,located upstream or downstream of the gene contained by the isolatednucleic acid molecule when found in the chromosome. In yet anotherembodiment, the isolated nucleic acid lacks one or more introns.Isolated nucleic acid molecules include sequences inserted intoplasmids, cosmids, artificial chromosomes, and the like. Thus, in aspecific embodiment, a recombinant nucleic acid is an isolated nucleicacid. An isolated protein may be associated with other proteins ornucleic acids, or both, with which it associates in the cell, or withcellular membranes if it is a membrane-associated protein. An isolatedorganelle, cell, or tissue is removed from the anatomical site in whichit is found in an organism. An isolated material may be, but need notbe, purified.

The term “purified” as used herein refers to material, such as a Gbanucleic acid or polypeptide, that has been isolated under conditionsthat reduce or eliminate unrelated materials, i.e., contaminants. Forexample, a purified protein is preferably substantially free of otherproteins or nucleic acids with which it is associated in a cell. As usedherein, the term “substantially free” is used operationally, in thecontext of analytical testing of the material. Preferably, purifiedmaterial substantially free of contaminants is at least 50% pure; morepreferably, at least 90% pure, and more preferably still at least 99%pure. Purity can be evaluated by chromatography, gel electrophoresis,immunoassay, composition analysis, biological assay, and other methodsknown in the art.

The term “host cell” means any cell of any organism that is selected,modified, transformed, grown, or used or manipulated in any way, for theproduction of a substance by the cell, for example the expression by thecell of a gene, a DNA or RNA sequence, a protein or an enzyme. Accordingto the present invention, the host cell is modified to express a mutantor wild-type lysosomal enzyme nucleic acid and polypeptide. Host cellscan further be used for screening or other assays. A “recombinant DNAmolecule” is a DNA molecule that has undergone a molecular biologicalmanipulation. Exemplary host cells for use in the present invention areHEK293 cells, COS cells, and CHO cells.

The polynucleotides herein may be flanked by natural regulatory(expression control) sequences, or may be associated with heterologoussequences, including promoters, internal ribosome entry sites (IRES) andother ribosome binding site sequences, enhancers, response elements,suppressors, signal sequences, polyadenylation sequences, introns, 5′-and 3′-non-coding regions, and the like. The nucleic acids may also bemodified by many means known in the art. Non-limiting examples of suchmodifications include methylation, “caps”, substitution of one or moreof the naturally occurring nucleotides with an analog, andinternucleotide modifications such as, for example, those with unchargedlinkages (e.g., methyl phosphonates, phosphotriesters,phosphoroamidates, carbamates, etc.) and with charged linkages (e.g.,phosphorothioates, phosphorodithioates, etc.). Polynucleotides maycontain one or more additional covalently linked moieties, such as, forexample, proteins (e,g, nucleases, toxins, antibodies, signal peptides,poly-L-lysine, etc.), intercalators (e.g., acridine, psoralen, etc.),chelators (e.g., metals, radioactive metals, iron, oxidative metals,etc.), and alkylators. The polynucleotides may be derivatized byformation of a methyl or ethyl phosphotriester or an alkylphosphoramidate linkage. Furthermore, the polynucleotides herein mayalso be modified with a label capable of providing a detectable signal,either directly or indirectly. Exemplary labels include radioisotopes,fluorescent molecules, biotin, and the like.

A “coding sequence” or a sequence “encoding” an expression product, suchas a RNA or polypeptide, is a nucleotide sequence that, when expressed,results in the production of that RNA or polypeptide, e.g., the Gbanucleotide sequence encodes an amino acid sequence for a Gba polypeptide(protein). A coding sequence for the protein may include a start codon(usually ATG) and a stop codon.

The term “gene”, also called a “structural gene” means a DNA sequencethat codes for or corresponds to a particular sequence of amino acidswhich comprise all or part of one or more lysosomal proteins, and may ormay not include regulatory DNA sequences, such as promoter sequences,which determine for example the conditions under which the gene isexpressed.

The terms “express” and “expression”, when used in the context ofproducing an amino acid sequence from a nucleic acid sequence, meansallowing or causing the information in a gene or DNA sequence to becomemanifest, for example producing a Gba protein by activating the cellularfunctions involved in transcription and translation of the correspondingGba gene or DNA sequence. A DNA sequence is expressed in or by a cell toform an “expression product” such as a Gba protein. The expressionproduct itself, e.g., the resulting protein, may also be said to be“expressed” by the cell. An expression product can be characterized asintracellular, extracellular or secreted. According to the presentinvention, the protein is expressed intracelluarly in neurons.

The term “intracellular” means something that is inside a cell. The term“extracellular” means something that is outside a cell. A substance is“secreted” by a cell if it appears in significant measure outside thecell, from somewhere on or inside the cell.

The term “heterologous” refers to a combination of elements notnaturally occurring in combination. For example, heterologous DNA refersto DNA not naturally located in the cell, or in a chromosomal site ofthe cell. Preferably, the heterologous

DNA includes a gene foreign to the cell. A heterologous expressionregulatory element is an element operatively associated with a differentgene than the one it is operatively associated with in nature. In thecontext of the present invention, a gene encoding a protein of interestis heterologous to the vector DNA in which it is inserted for cloning orexpression, and it is heterologous to a host cell containing such avector, in which it is expressed, e.g., an E. coli cell.

The term “transformation” refers to the process by which DNA, i.e. , anucleic acid encoding a lysosomal enzyme polypeptide, is introduced fromthe surrounding medium into a host cell.

The term “transduction” refers to the introduction of DNA, i.e., anucleic acid encoding a Gba polypeptide, into a prokaryotic host cell,e.g., into a prokaryotic host cell via a bacterial virus, orbacteriophage. A prokaryotic or eukaryotic host cell that receives andexpresses introduced DNA or RNA has been “transformed” or “transduced”and is a “transformant” or a “clone.” The DNA or RNA introduced into ahost cell can come from any source, including cells of the same genus orspecies as the host cell, or cells of a different genus or species, orsynthetic sequences.

The term “recombinantly engineered cell” refers to any prokaryotic oreukaryotic cell that has been manipulated to express or overexpress thenucleic acid of interest, i.e., a nucleic acid encoding a Gbapolypeptide, by any appropriate method, including transfection,transformation or transduction. This term also includes endogenousactivation of a nucleic acid in a cell that does not normally expressthat gene product or that expresses the gene product at a sub-optimallevel.

The term “transfection” means the introduction of a foreign” (i.e. ,extrinsic or extracellular) nucleic acid into a cell. The “foreign”nucleic acid includes a gene, DNA or RNA sequence to a host cell, sothat the host cell will replicate the DNA and express the introducedgene or sequence to produce a desired substance, typically a protein orenzyme coded by the introduced gene or sequence. The introduced gene,i.e. , a nucleic acid encoding a Gba polypeptide, or sequence may alsobe called a “cloned” gene or sequence, may include regulatory or controlsequences, such as start, stop, promoter, signal, secretion, or othersequences used by a cell's genetic machinery. The gene or sequence mayinclude nonfunctional sequences or sequences with no known function. DNAmay be introduced either as an extrachromosomal element or bychromosomal integration or a host cell that receives and expressesintroduced DNA or RNA.

Depending on the host cell used, transformation/transfection is doneusing standard techniques appropriate to such cells. The calciumtreatment employing calcium chloride, as described in section 1.82 ofSambrook et al., 1989 supra, is generally used for bacterial cells thatcontain substantial cell-wall barriers. Another method fortransformation employs polyethylene glycol/DMSO, as described in Chungand Miller (Nucleic Acids Res. 1988, 16: 3580). Yet another method isthe use of the technique termed electroporation. Alternatively, where aviral vector is used, the host cells can be infected by the viruscontaining the gene of interest.

The terms “vector”, “cloning vector” and “expression vector” mean thevehicle by which a DNA or RNA sequence (e.g., a Gba gene) can beintroduced into a host cell, so as to transform the host and promoteexpression (e.g., transcription and translation) of the introducedsequence. Vectors include plasmids, phages, viruses, etc.; they arediscussed in greater detail below.

Vectors typically comprise the DNA of a transmissible agent, into whichforeign DNA is inserted. A common way to insert one segment of DNA intoanother segment of DNA involves the use of enzymes called restrictionenzymes that cleave DNA at specific sites (specific groups ofnucleotides) called restriction sites. A “cassette” refers to a DNAcoding sequence or segment of DNA that codes for an expression productthat can be inserted into a vector at defined restriction sites. Thecassette restriction sites are designed to ensure insertion of thecassette in the proper reading frame. Generally, foreign DNA is insertedat one or more restriction sites of the vector DNA, and then is carriedby the vector into a host cell along with the transmissible vector DNA.A segment or sequence of DNA having inserted or added DNA, such as anexpression vector, can also be called a “DNA construct.” A common typeof vector is a “plasmid”, which generally is a self-contained moleculeof double-stranded DNA, usually of bacterial origin, that can readilyaccept additional (foreign) DNA and which can readily introduced into asuitable host cell. A plasmid vector often contains coding DNA andpromoter DNA and has one or more restriction sites suitable forinserting foreign DNA. Coding DNA is a DNA sequence that encodes aparticular amino acid sequence for a particular protein or enzyme.Promoter DNA is a DNA sequence which initiates, regulates, or otherwisemediates or controls the expression of the coding DNA. Promoter DNA andcoding DNA may be from the same gene or from different genes, and may befrom the same or different organisms.

A large number of vectors, including plasmid and fungal vectors, havebeen described for replication and/or expression in a variety ofeukaryotic and prokaryotic hosts. Non-limiting examples include pKKplasmids (Clonetech), pUC plasmids, pET plasmids (Novagen, Inc.,Madison, Wis.), pRSET or pREP plasmids (Invitrogen, San Diego, Calif.),or pMAL plasmids (New England Siolabs, Beverly, Mass.), pCXN and manyappropriate host cells, using methods disclosed or cited herein orotherwise known to those skilled in the relevant art. Recombinantcloning vectors will often include one or more replication systems forcloning or expression, one or more markers for selection in the host,e.g., antibiotic resistance, and one or more expression cassettes.

A wide variety of host/expression vector combinations (i.e., expressionsystems) may be employed in expressing the proteins of interest. Usefulexpression vectors, for example, may consist of segments of chromosomal,non-chromosomal and synthetic DNA sequences. Suitable vectors includeknown bacterial plasmids, e.g., E. coil plasmids col E1, pCR1, pBR322,pMal-C2, pET, pGEX (Smith et al., Gene 67:31-40, 1988), pMB9 and theirderivatives, plasmids such as RP4; phage DNAS, e.g., the numerousderivatives of phage 1, e.g., NM989, and other phage DNA, e.g., M13 andfilamentous single stranded phage DNA; yeast plasmids such as the 2mplasmid or derivatives thereof; vectors derived from combinations ofplasmids and phage DNAs, such as plasmids that have been modified toemploy phage DNA or other expression control sequences; and the like.Another common expression system uses insect host cells and baculovirusvectors.

Exemplary expression vectors commercially available for use in mammaliancells include pMEP4, pCEP4, pLXSN, PXT1, pcDNA3 series, pcDNA4 series,pCMV-Script, pCMV-Tag and other CMV-based vectors, pVP22, pVAX1, pUB6.For transfection of mammalian cells, viral vectors includeadeno-associated viral vectors, pox viruses, and retroviruses. Mammalianexpression vectors are routine and well known in the art.

The host cells can inherently also harbor the polypeptide of interest,eg., Gba. For heterologous polypeptides such as Gba, the heterologousnucleic acid (e.g., cDNA) is suitably inserted into a replicable vectorfor expression in the culture medium under the control of a suitablepromoter. As noted above, many vectors are available for this purpose,and selection of the appropriate vector will depend mainly on the sizeof the nucleic acid to be inserted into the vector and the particularhost cell to be transformed with the vector. Each vector containsvarious components depending on its function (amplification of DNA orexpression of DNA) and the particular host cell with which it iscompatible. The vector components for bacterial transformation generallyinclude, but are not limited to, one or more of the following: a signalsequence, an origin of replication, one or more marker genes, and apromoter.

The DNA encoding the Gba polypeptide may be expressed not only directly,but also as a fusion with another polypeptide, preferably a signalsequence or other polypeptide having a specific cleavage site at theN-terminus of the mature polypeptide. In general, the signal sequencemay be a component of the vector, or it may be a part of the polypeptideDNA that is inserted into the vector. The heterologous signal sequenceselected should be one that is recognized and processed (i.e., cleavedby a signal peptidase) by the host cell. For bacterial host cells thatdo not recognize and process the native polypeptide signal sequence, thesignal sequence is substituted by a bacterial signal sequence selected,for example, from the group consisting of the alkaline phosphatase,penicillinase, 1 pp, or heat-stable enterotoxin II leaders.

Both expression and cloning vectors contain a nucleic acid sequence thatenables the vector to replicate in one or more selected host cells.Generally, in cloning vectors this sequence is one that enables thevector to replicate independently of the host chromosomal DNA, andincludes origins of replication or autonomously replicating sequences.Such sequences are well known for a variety of bacteria. The origin ofreplication from the plasmid pBR322 is suitable for most Gram-negativebacteria.

Expression and cloning vectors also generally contain a selection gene,also termed a selectable marker. This gene encodes a protein necessaryfor the survival or growth of transformed host cells grown in aselective culture medium. Host cells not transformed with the vectorcontaining the selection gene will not survive in the culture medium.Typical selection genes encode proteins that (a) confer resistance toantibiotics or other toxins, e.g., ampicillin, neomycin, methotrexate,or tetracycline; (b) complement auxotrophic deficiencies; or (c) supplycritical nutrients not available from complex media. One example of aselection scheme utilizes a drug to arrest growth of a host cell. Thosecells that are successfully transformed with a heterologous gene producea protein conferring drug resistance and thus survive the selectionregimen.

The expression vector for producing a heterologous polypeptide alsocontains an inducible promoter that is recognized by the host organismand is operably linked to the nucleic acid encoding the polypeptide ofinterest.

A “promoter sequence” is a DNA regulatory region capable of binding RNApolyrnerase in a cell and initiating transcription of a downstream (3′direction) coding sequence. For purposes of defining the presentinvention, the promoter sequence is bounded at its 3′ terminus by thetranscription initiation site and extends upstream (5′ direction) toinclude the minimum number of bases or elements necessary to initiatetranscription at levels detectable above background. Within the promotersequence will be found a transcription initiation site, as well asprotein binding domains (consensus sequences) responsible for thebinding of RNA polymerase.

A coding sequence is “under the control” of or “operatively associatedwith” transcriptional and translational control sequences in a cell whenRNA polymerase transcribes the coding sequence into mRNA, which is thentrans-RNA spliced (if it contains introns) and translated into theprotein encoded by the coding sequence.

Construction of suitable vectors containing one or more of the abovelisted components employs standard ligation techniques. Isolatedplasmids or DNA fragments are cleaved, tailored, and religated in theform desired to generate the plasmids required.

For analysis to confirm correct sequences in plasmids constructed, theligation mixtures are used to transform bacterial strains, andsuccessful transformants are selected by ampicillin or tetracyclineresistance where appropriate. Plasmids from the transformants areprepared, analyzed by restriction endonuclease digestion, andiorsequenced by the method of Sanger et al., Proc. Nat. Acad. Sci. USA.1977, 74:5463-5467 or Messing et al., Nucleic Acids Res. 1981, 9: 309),or by the method of Maxam et al. (Methods in Enzymology 1980, 65: 499).Host cells are transformed with the above-described expression vectorsand cultured in conventional nutrient media modified as appropriate forthe promoter utilized.

Chemical Definitions

The term ‘alkyl’ refers to a straight or branched C₁-C₂₀ hydrocarbongroup consisting solely of carbon and hydrogen atoms, containing nounsaturation, and which is attached to the rest of the molecule by asingle bond, e.g., methyl, ethyl, n-propyl, 1-methylethyl (isopropyl),n-butyl, n-pentyl, 1,1-dimethylethyl (t-butyl). The alkyls used hereinare preferably C₁-C₈ alkyls.

The term “alkenyl” refers to a C₂-C₂₀ aliphatic hydrocarbon groupcontaining at least one carbon-carbon double bond and which may be astraight or branched chain, e.g., ethenyl, 1-propenyl, 2-propenyl(allyl), iso-propenyl, 2-methyl-1-propenyl, 1-butenyl, 2-butenyl.

The term “cycloalkyl” denotes an unsaturated, non-aromatic mono- ormulticyclic hydrocarbon ring system such as cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl. Examples of multicyclic cycloalkyl groupsinclude perhydronapththyl, adarnantyl and norbornyl groups bridgedcyclic group or sprirobicyclic groups, e.g., Spiro (4,4) non-2-yl.

The term “cycloalkalkyl” refers to a cycloalkyl as defined abovedirectly attached to an alkyl group as defined above, that results inthe creation of a stable structure such as cyclopropylmethyl,cyclobutylethyl, cyclopentylethyl.

The term “alkyl ether” refers to an alkyl group or cycloalkyl group asdefined above having at least one oxygen incorporated into the alkylchain, e.g., methyl ethyl ether, diethyl ether, tetrahydrofuran.

The term “alkyl amine” refers to an alkyl group or a cycloalkyl group asdefined above having at least one nitrogen atom, e.g., n-butyl amine andtetrahydrooxazine.

The term “aryl” refers to aromatic radicals having in the range of about6 to about 14 carbon atoms such as phenyl, naphthyl, tetrahydronapthyl,indanyl, biphenyl.

The term “arylalkyl” refers to an aryl group as defined above directlybonded to an alkyl group as defined above, e.g., —CH₂C₆H₅, and—C₂H₄C₆H₅.

The term “heterocyclic” refers to a stable 3- to 15-membered ringradical which consists of carbon atoms and from one to five heteroatomsselected from the group consisting of nitrogen, phosphorus, oxygen andsulfur. For purposes of this invention, the heterocyclic ring radicalmay be a monocyclic, bicyclic or tricyclic ring system, which mayinclude fused, bridged or Spiro ring systems, and the nitrogen,phosphorus, carbon, oxygen or sulfur atoms in the heterocyclic ringradical may be optionally oxidized to various oxidation states. Inaddition, the nitrogen atom may be optionally quaternized; and the ringradical may be partially or fully saturated (i.e., heteroaromatic orheteroaryl aromatic), Examples of such heterocyclic ring radicalsinclude, but are not limited to, azetidinyl, acridinyl, benzodioxolyl,benzodioxanyl, benzofurnyl, carbazolyl, cinnolinyl, dioxolanyl,indolizinyl, naphthyridinyl, perhydroazepinyl, phenazinyl,phenothiazinyl, phenoxazinyl, phthalazinyl, pyridyl, pteridinyl,purinyl, quinazolinyl, quinoxalinyl, quinolinyl, isoquinolinyl,tetrazoyl, imidazolyl, tetrahydroisouinolyl, piperidinyl, piperazinyl,2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolidinyl, 2-oxoazepinyl,azepinyl, pyrrolyl, 4-piperidonyl, pyrrolidinyl, pyrazinyl, pyrimidinyl,pyridazinyl, oxazolyl, oxazolinyl, oxasolidinyl, triazolyl, indanyl,isoxazolyl, isoxasolidinyl, morpholinyl, thiazolyl, thiazolinyl,thiazolidinyl, isothiazolyl, quinuclidinyl, isothiazolidinyl, indolyl,isoindolyl, indolinyl, isoindolinyl, octahydroindolyl,octahydroisoindolyl, quinolyl, isoquinolyl, decahydroisoquinolyl,benzimidazolyl, thiadiazolyl, benzopyranyl, benzothiazolyl,benzooxazolyl, furyl, tetrahydrofurtyl, tetrahydropyranyl, thienyl,benzothienyl, thiamorpholinyl, thiamorpholinyl sulfoxide thiamorpholinylsulfone, dioxaphospholanyl, oxadiazolyl, chromanyl, isochromanyl.

The heterocyclic ring radical may be attached to the main structure atany heteroatom or carbon atom that results in the creation of a stablestructure.

The term “heteroaryl” refers to a heterocyclic ring wherein the ring isaromatic.

The term “heteroarylalkyl” refers to heteroaryl ring radical as definedabove directly bonded to alkyl group. The heteroarylalkyl radical may beattached to the main structure at any carbon atom from alkyl group thatresults in the creation of a stable structure.

The term “heterocyclyl” refers to a heterocylic ring radical as definedabove. The heterocyclyl ring radical may be attached to the mainstructure at any heteroatom or carbon atom that results in the creationof a stable structure.

The term “heterocyclylalkyl” refers to a heterocylic ring radical asdefined above directly bonded to alkyl group. The heterocyclylalkylradical may be attached to the main structure at carbon atom in thealkyl group that results in the creation of a stable structure.

The substituents in the ‘substituted alkyl’, ‘substituted alkenyl’‘substituted alkynyl’ ‘substituted cycloalkyl’ ‘substitutedcycloalkalkyl’ ‘substituted cyclocalkenyl’ ‘substituted arylalkyl’‘substituted aryl’ ‘substituted heterocyclic ring’, ‘substitutedheteroaryl ring,’ ‘substituted heteroarylalkyl’, or ‘substitutedheterocyclylalkyl ring’, may be the same or different with one or moreselected from the groups hydrogen, hydroxy, halogen, carboxyl, cyano,amino, nitro; oxo (═O), thio (═S), or optionally substituted groupsselected from alkyl, alkoxy, alkenyl, alkynyl, aryl, arylalkyl,cycloalkyl, aryl, heteroaryl, heteroarylalkyl, heterocyclic ring,—COOR^(x), —C(O)R^(c), —C(S)R^(x), —C(O)NR^(x)R^(y), —C(O)ONR^(x)R^(y),—NR^(x)CONR^(y)R^(z), —N(R^(x))SOR^(y), —N(R^(x))SO₂R^(y),—(═N-N(R^(x))R^(y)), —NR^(x)C(O)OR^(y), —NR^(x)R^(y), —NR^(x)C(O)R^(y),—NR^(x)C(S)R^(y), —NR^(x)C(S)NR^(y)R^(z), —SONR^(x)R^(y),—SO₂NR^(x)R^(y)—, —OR^(x), —OR^(x)C(O)NR^(y)R^(z), —OR^(x)C(O)OR^(y)—,—OC(O)R^(x), —OC(O)NR^(x)R^(y), —R^(x)NR^(y)R^(z), —R^(x)R^(y)R^(z),—R^(x)CF₃, —R^(x)NR^(y)C(O)R^(z), —R^(x)OR^(y), —R^(x)C(O)OR^(y),—R^(x)C(O)NR^(y)R^(z), —R^(x)C(O)R^(x), —R^(x)OC(O)R^(y), —SR^(x),—SOR^(x), —SO₂R^(x), —ONO₂, wherein R^(x), R^(y) and R^(z) in each ofthe above groups can be hydrogen atom, substituted or unsubstitutedalkyl, haloalkyl, substituted or unsubstituted arylalkyl, substituted orunsubstituted aryl, substituted or unsubstituted cycloalkyl, substitutedor unsubstituted cycloalkalkyl substituted or unsubstituted heterocyclicring, substituted or unsubstituted heterocyclylalkyl, substituted orunsubstituted heteroaryl or substituted or unsubstitutedheteroarylalkyl.

The term “halogen” refers to radicals of fluorine, chlorine, bromine andiodine.

Toxic Gain of Function

In one particular embodiment, the invention relates to the use ofspecific pharmacologic chaperones for a lysosomal enzyme to increase thelevel of appropriate protein trafficking and decrease the level ofmutant enzyme accumulation. This in turn, can be used to treatneurological conditions associated with a mutation or mutations in theenzyme, including forms of lysosomal storages diseases in which themutation on one or both alleles yields enzymes which are conformationalmutations but which also have mutations in functional domains,abrogating enzyme activity. This embodiment is exemplified herein by theeffect of a specific pharmacological chaperone on a mutant Gba found ina neurological form of Gaucher disease where there was no functional Gbapresent. The chaperone increased the level of Gba protein traffickingfrom the ER, and restored proper ubiquitination of the mutant protein.This effect was not foreseeable from the prior work on ASSC rescue ofprotein function.

Protein aggregation, such as mutant Gba accumulation, in the CNS isparticularly dire since neurons are unable to regenerate followingneurodegeneration or apoptosis that arises from neuronal stressassociated with the toxic accumulation. Thus, the presence ofhomozyogous or heterozygous mutations is sufficient to cause mutantprotein aggregation or accumulation in neurons and cell stress,ultimately leading to cell death. Numerous reports have been publishedlinking protein aggregation in the CNS to pathology.

Therefore, in one embodiment, the present invention is based, on theconcept that CNS pathology in lysosomal storage diseases, and otherneurological disorders associated with mutations in lysosomal enzymescan be explained, in part, by toxic accumulation of mutant, misfoldedenzymes in neurons, and that a specific pharmacological chaperoneapproach can reverse this effect. The toxic effect also is dependentupon the protein's function, the effects of the mutation on theprotein's function and stability, and whether loss or reduction ofprotein function is more or less deleterious than the toxic affects ofprotein accumulation and/or aggregation. Accordingly, increasingtrafficking of the protein from the ER using a specific pharmacologicalchaperone can alleviate disease pathology by reducing the toxic effectsof protein accumulation/aggregation, even without necessarily restoringprotein function.

It follows that specific pharmacological chaperones could potentially beused to treat any disease in which a significant contributor to diseasepathology is toxic accumulation of protein and/or protein aggregation,including that associated with neurodegenerative diseases, especiallylysosomal storage diseases with neurological involvement such as Gaucherdisease, and other neurological risks factors, disorders, or conditionsassociated with mutations in lysosomal enzymes, such as Parkinson'sdisease.

As indicated above, other types of neurological disease that may beassociated with a dysfunctional lysosomal enzyme and thus may be treatedby pharmacological chaperones are Alzheimer's, Amyotrophic LateralSclerosis, Canavan's, Creutzfeldt-Jakob, Huntington's, MultipleSclerosis, Pick's Disease, and Spinocerebellar Atrophy.

Accordingly, a treatment method that increases mutant enzyme transportfrom the ER, and/or increases enzyme activity, is beneficial inmitigating the neuronopathic effects associated with the lysosomalstorage disease or other associated neurological diseases that arelinked with mutations in lysosomal enzymes. Even in the absence ofincreasing enzyme activity (i.e., restoring loss of function), andreducing the accumulation of substrate, proper trafficking of mutantenzyme has beneficial effects on the neuron such as (i) alleviating cellstress on the ubiquitin/proteasome degradation pathway for normalproteins; or (ii) reducing the unfolded protein response caused by ERstress, thus improving pathologies such as e.g., α-synuclein aggregationin Parkinson's patients having mutations in Gba. Support for theseeffects is provided directly below.

Cell stress. It is well established that accumulation or aggregation ofnumerous misfolded proteins in a cell leads to cell stress. This stressis sometimes correlated with increased amounts of polyubiquitin, a cell“stress” protein. Ubiquitin-protein conjugates have revealed thatubiquitin is a component of many of the filamentous inclusion bodiescharacteristic of neurodegenerative diseases, suggesting activation of acommon neuronal response in this type of disease process (Lowe et al.,Neuropathol Appl Neurobiol. 1990; 16: 281-91). For example, geneticstudies, including identification of mutations in genes associated withfamilial Parkinson's (including α-synuclein), and the presence ofproteinaceous cytoplasmic inclusions in spared dopaminergic nigralneurons in sporadic cases of Parkinson's have suggested an importantrole for ubiquitin-proteasome system and aberrant protein degradation(Betarbet et al., Exp Neural. 2005;191 Suppl 1:S17-27).

In addition, in vivo and in vitro studies have linked aggregatedα-synuclein and oxidative stress to a compromised ubiquitin-proteasomesystem and Parkinson's disease pathogenesis. Moreover, structural andfunctional defects in 26/20S proteasomes with accumulation andaggregation of potentially cytotoxic abnormal proteins have beenidentified in the substantia nigra pars compacta of patients withsporadic Parkinson's disease (McKnaught et al., Ann Neural, 2003; 53Suppl 3:S73-84). Specifically, mutations in a-synuclein that cause theprotein to misfold and resist proteasomal degradation cause familialParkinson's. Thus, a defect in protein handling appears to be a commonfactor in sporadic and the various familial forms of PD. This sameconclusion was drawn from experiments in which a combination of aproteasome inhibitor with agents that induce protein misfolding wereadded to a culture of dopaminergic neurons (Mytilineou et al., J NeuralTransm. 2004; 111(10-11):1237-51). Preferential loss of dopamine neuronsand cell death is markedly increased when the two are combined.

Further, it has been reported that ubiquitinated protein aggregates werefound in patient cells for some lysosomal storage diseases, includingGaucher disease (Asmarina et al., Eur. J. Biochem. 2003; Supplement 1;abstract no. P3.7-08). These cells also displayed altered geneexpression patterns for genes related to the ubiquitin/proteasomepathway.

An alternate theory for disruptions in neuronal homeostasis in LSDs withCNS involvement is due to suppression of the ubiquitin/proteasomepathway by the accumulated enzymes (Rocca et al., Molecular Biology ofthe Cell. 2001; 12: 1293-1301). For example, it has been found that oneof the mechanisms of toxicity associated with α-synuclein aggregation isproteasomal inhibition, which occurs in many neurodegenerativeprocesses. Specifically, it was shown that aggregated a-synucleininhibits proteasomal function by interacting with S6′, a subunit of theproteasome (Snyder et al., J Mol Neurosci. 2004;24(3):425-42).Proteasomal function is decreased in brains of subjects with Parkinson'sdisease as well as in brains from individuals and animals lackingparkin, which is an E3 ubiquitin ligase and part of the ubiquitinproteasomal system. Protein aggregation and associated proteasomalinhibition has also been linked to inflammation (Li et al., int. J.Biochem. Cell Biol. 2003; 35: 547-552). It has been proposed that animbalance between molecular chaperones and damaged/denatured/misfoldedproteins, leading to accumulation of the latter, can result insenescence, inhibition of the proteasome (leading to apoptosis), ornecrosis, depending on the severity of the imbalance (Soti et al., AgingCell. 2003; 2: 39-45). This hypothesis is referred to as the “toxicprotein accumulation hypothesis.” Since a-synuclein monomers are thoughtto be degraded by the proteasomes and oligomer formation isconcentration dependent, this could lead to an accumulation andoligomerization of α-synuclein. The accumulation of both mutant Gba anda-synuclein (the latter due to loss of Gba activity) would exacerbatethis effect on the proteasomes, and deficient Gba may also impair anyincrease in the autophagic response by lysosomes that occurs tocompensate for the deficiency of the proteasome degradation pathway.

ER stress. In addition to the above-referenced discussion, continuedaccumulation of misfolded proteins in the lumen of the ER creates an ERstress response, which, in turn, elicits the “unfolded protein response”(UPR). The UPR is a quality control cell stress response that resultsfrom inhibition of protein synthesis, such as by oxidative stress, orretention of mutant proteins in the ER that are unable to fold. Withoutthis response, the ER becomes engorged with misfolded, unstable proteinswhich can result in cell death via apoptosis (Gow et al., NeuroMolecularMed. 2003; 4: 73-94).

It has also been shown that Gba interacts with the Rhyanodine receptorin the ER to disturb Ca2+ homeostasis, leading to impaired proteinfolding and a UPR, ER-stress induced apoptosis andmitochondrial-directed cell death due to an increase in cytosolic Ca2+(Korkotian et al., J Biol Chem. 1999. 274(31): 21673-8; Lloyd-Evans etal., J Biol Chem. 2003. 278(26): 23594-9; Pelled et al., Neurobiol Dis.2005. 18(1): 83-8).

Autophagy. In addition to degrading lipids, lysosomes are responsiblefor degrading aggregated proteins (discussed further below). Thisprocess, called autophagy, is an intracellular bulk degradation processthrough which a portion of the cytoplasm is delivered to lysosomes to bedegraded by lysosomal enzymes. Such enzymes include proteases(cathepsins) which cleave peptide bonds, phosphatases, which removecovalently bound phosphates, nucleases, which cleave DNA/RNA, lipases,which cleave lipid molecules, and carbohydrate-cleaving enzymes.Aggregated proteins, including mutated lysosomal enzymes, can causeactivation of a conspicuous autophagic response leading to long-lastingdegenerative changes in neurons. Many neurons in CNS disorders,including amyotrophic lateral sclerosis (ALS), exhibit irregularvesicular trafficking and autophagic responses.

It is possible that excessive autophagic-lysosomal vacuolation can causeneuronal death. Over activation of the autophagic response, especiallyin combination with inhibition of the proteasome pathway as acompensatory mechanism, by accumulated mutant proteins is one hypothesisfor a link between accumulated mutant lysosomal enzymes andneurodegeneration, especially in Alzheimer's disease.

Pathologic Loss of Function

In addition to restoring proper trafficking of lysosomal enzymes,specific pharmacological chaperone restoration of mutant enzyme activitywill be beneficial in patients harboring a destabilizing mutation(s) inone or both alleles which reduces the amount of functional enzyme (e.g.,Gba) at its native location (e.g., the lysosome) due to inefficientfolding and trafficking. Even a small loss of function can lead topathologies such as substrate accumulation or aggregation, which canresult in seeding of other pathologic aggregates.

Therefore, in one embodiment, the present invention provides methods forimproving neurological disorders associated with mutant lysosomal enzymeproteins by increasing reduced activity of the enzymes which will, inturn, (i) increase lysosomal degradation of substrates, aggregatedproteins or fragments; (ii) decrease neuronal apoptosis or necrosis; and(iii) prevent alteration of the phospholipid “balance” in cell membranes(discussed directly below).

Possible explanations postulated to explain the neuronal loss orneuropathy in Gaucher disease can be explained by the loss of Gbaactivity associated with the mutations. Loss of activity causes theaccumulation of ceramide, such as the GluCer, in cells with deficientGba. This has been shown to cause apoptosis in cultured hippocampalCA2-4 neuron cells, due to an increase in intracellular calcium. and anincrease in sensitivity to calcium-mediated cell death. Dopaminergicneurons have also been shown to undergo apoptosis after ceramide-induceddamage.

Second, high levels of the toxic compound glucosylsphingosine, also asubstrate of Gba, have been observed in organs from lethal null alleleGaucher mice. Glucosylsphingosine also is elevated in tissues frompatients having all three types of Gaucher disease. Although brainlevels are elevated only in those patients with neuronal involvementusing current methods of detection (Sidransky, Mol. Gen. Metabol. 2004;83: 6-15), small amounts of accumulation not detectable using currentmethods could still affect protein folding and also impair proteintrafficking by affecting lipid raft composition (discussed infra).

Third, membrane phospholipid content affects the activity of Gba incells. Namely, negatively charged phospholipids enhance Gba activity,and positively charged phospholipids such as phosphotidyicholine (PC) donot. Therefore, a mechanism where decreased Gba in Gaucher diseaseactivates an enzyme involved in the synthesis of PC, thereby increasingPC, may cause a further reduction in Gba (Wong et al., supra). Inaddition, elevated ceramide may hinder axonal transport of α-synuclein,favoring aggregation and Lewy body formation. Neurons presumably requirea-synuclein for function. Since a-synuclein binds PC poorly, axonaltransport vesicles that are comprised primarily of PC may not be asefficient as vesicles comprised of acidic phospholipids (Wong et al.,supra).

Further, as discussed above, lysosomes are involved in clearingaggregates involved in numerous CNS disorders by autophagy. Autophagy isparticularly relevant in neurons, since loss of autophagy causesneurodegeneration even in the absence of any disease-associated mutantproteins (Hara et al., Nature. online publication Apr. 19, 2006).Induction of the lysosomal autophagic system, in a protective effort toeliminate altered intracellular components occurs during oxidativestress (Kiffin et al., Antioxid Redox Signal. 2006;8(1-2): 152-62).

As one example, α-synuclein oligomers. One group reported an interactionbetween glucosylceramide containing gangliosides and α-synuclein inlysosomes in human brain homongenates (Schlossmacher et al., New Eng JMed. 2005; 352: 730). In Gaucher patients with Parkinson's, Gbacolocalized with α-synuclein in Lewy bodies (Wong et al., Mol. Genet.Metabol. 2004; 38: 192-207). These results support that processing ofa-synuclein occurs within lysosornes, and provides a biochemical linkbetween decreased Gba activity and synucleinopathy in Parkinson'sdisease.

In addition, autophagy is essential for the elimination of aggregatedforms of mutant huntingtin and ataxin-1 from the cytoplasmic compartment(Iwata et al., Proc Natl Aced Sci USA. 2005;102(37): 13135-40).Autophagy also plays the major role in clearing of cells from proteinaggregates in Amyotrophic Lateral Sclerosis, Alzheimer's disease,Parkinson's disease, Huntington Disease and other polyglutamineexpansion disorders eriin et al., Int J Hyperthermia. 2005; 21(5):403-19). Thus, deficiencies in lysosomal hydrolases would adverselyaffect the autophagic response to toxic accumulation of proteins(including accumulated lysosomal proteins themselves)

Substrate accumulation and endocytic trafficking defects. Accumulationof cellular substrates, such as the sphingolipids and cholesterol inlysosomal diseases, especially those involving the CNS, has beenassociated with disruptions in endocytic trafficking of proteins andlipids. This may occur because of the disruption of rab (ras in thebrain) proteins, which are membrane associated proteins that localize todiscrete subcellular compartments and are associated with proteintrafficking. The rab disruption causes sequestering bymembrane-associated proteins into “lipid rafts.” Lipid rafts aremembrane microdomains enriched in sphingolipids (sphingomyelin andphosphotidyicholine) and cholesterol. They have been suggested to serveas platforms for various cellular events, including signaling andmembrane trafficking. In particular, lipid rafts stabilize theassociation of GPI-anchored proteins within the ER membrane and aredirectly involved in protein conformation and also direct the lipids orlipid-associated proteins entering the cell to the appropriatecompartment via endosomes. Therefore, the accumulation of lipid rafts inmembranes of endosomes and lysosomes in e.g., lysosomal storagediseases, due to decreased lipid hydrolase activity, could alterintracellular sorting of glycosphingolipids (which are alreadyaccumulated), and lipid-associated proteins which enter the cell (Paganoet al., Philos Trans R Soc Lond B Biol Sci. 2003; 358: 885-91).

This mis-sorting hypothesis is supported by recent findings inmucopolysaccharidoses (MPSs), where it was demonstrated that twodifferent accumulated substrates, G_(M2) and G_(M3) gangliosides,accumulated in the same neurons, but were consistently located inseparate populations of cytoplasmic vesicles (McGlynn et al., CompNeurol. 2004; 480: 415-26). These authors hypothesized thatco-sequestration in individual neurons suggests the presence of defectsin the composition, trafficking, and/or recycling of lipid raftcomponents, leading to new mechanisms to explain neuronal dysfunction inMPS disorders.

Studies of mouse models for Gaucher disease also suggest that reducedGba activity more generally disrupts glycosphingolipid catabolismleading to accumulation of more complex species (gangliosides).Accumulation of gangliosides can results in dystonia and parkinsonism inhumans (Rote et al., Movement Disorders. 2005; 20(10): 1366-1369). Amouse model that accumulates G ganglioside also accumulated α-synucleinsynuclein (Suzuki et al., Neuroreport, 2003;14(4): 551-4. Suchaccumulation of gangliosides can also lead to α-synuclein accumulation,as well as neuronal death through the UPR pathways (Lee et al., J BiolChem. 2002. 277(1): 671-8). Further, as recited above, is has been shownthat sphingolipids can function as a seed for the formation ofα-synuclein aggregates.

These mechanisms of neurotoxicity as a result of accumulation of lipidspartially can explain the neuropathology of Gaucher disease, since thereis a loose correlation between Gba activity and Gaucher diseaseseverity. This correlation works to differentiate the three majordisease types (I-III), although there is overlap and the correlation isweak within the individual types. Patients who are heterozygous normalfor Gba do not experience significant accumulation of lipids, becausethere is some amount of active Gba produced by the normal allele.However, even accumulation of small amounts of GluCer can disrupt ERcalcium homeostasis and impair protein folding, (described above), orpossibly even seed α-synuclein aggregation by some mechanism.

In view of the foregoing, the use of specific pharmacological chaperonesaccording to the present invention is advantageous over enzymereplacement therapy (ERT) and substrate reduction therapy (SRT), sincethe former must be administered directly into the brain via a catheter,and since neither address the problems of toxic accumulation of themutant lysosomal enzymes themselves, i.e., mutant Gba. Therefore, thesetreatments are less effective than a treatment than can reduce mutantprotein accumulation, or enhance and/or restore protein function(thereby reducing substrate accumulation) or both.

Mutant Lysosomal Enzymes and Specific Pharmacological Chaperones

Following is a table which lists lysosomal enzymes and specificpharmacological chaperones for those lysosomal enzymes which can be usedto treat individuals having mutations in the enzymes and who have aresultant neurological condition or disorder, or are at risk ofdeveloping a neurological condition or disorder.

LYSOSOMAL ENZYME SPECIFIC PHARMACOLOGICAL CHAPERONE α-Glucosidase1-deoxynojirimycin (DNJ) GenBask Accession No. Y00839 α-homonojirimycincastanospermine Acid β-Glucosidase (glucocerebrosidase) isofagomineGenBank Accession No. J03059 C-benzyl isofagomine and derivativesN-alkyl (C9-12)-DNJ Glucoimidazole (and derivatives) C-alkyl-IFG (andderivatives) N-alkyl-β-valeinamines Fluphenozine calystegines A₃, B₁, B₂and C₁ α-Galactosidase A 1-deoxygalactonojirimycin (DGJ) GenBankAccession No. NM000169 α-allo-homonojirimycin α-galacto-homonojirimycinβ-1-C-butyl-deoxynojirimycin calystegines A₂ and B₂ N-methylcalystegines A₂ and B₂ Acid β-Galactosidass = GenBank Accession No.M34423 Galactocerebrosidase (Acid β-Galactosidase) 4-epi-isofagomineGenBank Accession No. D25283 1-deoxygalactonojirimycin Acidα-Mannosidase 1-deoxymannojirimycin GenBank Accession No. U68567Swainsonine Mannostatin A Acid β-Mannosidase 2-hydroxy-isofagomineGenBank Accession No. U60337 Acid α-L-fucosidase 1-deoxyfuconojirimycinGenBank Accession No. NM_000147 β-homofuconojirimycin2,5-imino-1,2,5-trideoxy-L-glucitol 2,5-deoxy-2,5-imino-D-fucitol2,5-imino-1,2,5-trideoxy-D-altritol α-N-Acetylglucosaminidase1,2-dideoxy-2-N-acetamido-nojirimycin GenBank Accession No. U40846α-N-Acetylgalactosaminidase 1,2-dideoxy-2-N-acetamido-galactonojirimycinGenBank Accession No. M62783 β-Hexosaminidase A2-N-acetylamino-isofagomine GenBank Accession No. NM_0005201,2-dideoxy-2-acetamido-nojirimycin nagstain β-Hexosaminidase B2-N-acetamido-isofagomine GenBank Accession No. NM_0005211,2-dideoxy-2-acetamido-nojirimycin nagstain α-L-Iduronidase1-deoxyiduronojirimycin GenBank Accession No. NM_0002032-carboxy-3,4,5-trideoxypiperidine β-Glucuronidase 6-carboxy-isofagomineGenBank Acccssion No. NM_000181 2-carboxy-3,4,5-trideoxypiperidineSialidase 2,6-dideoxy-2,6, imino-sialic acid GenBank Accession No.U84246 Siastatin B Iduronate sulfatase 2,5-anhydromannitol-6-sulphateGenBank Accession No. AF_011889 Acid sphingomyelinase desipramine,phosphatidylinositol-4,5-diphosphate GenBank Accession No. M59916

In one specific embodiment, following are some specific pharmacologicalchaperones contemplated by this invention which can be used for treatingneurological risk factors, conditions or disorders in which Gba ismutated. Also exemplified are Gba mutations contemplated to be “rescued”by the chaperones.

Gba mutations. The presence of Gba point mutation N370S on at least oneallele (heterozygotes) is almost universally associated with type 1Gaucher disease (Cox, supra). N370S homozygosity is associated with aless severe phenotype than Gba null/N370S heterozygosity (N370S/null),likely due to the residual Gba activity of the homozygotes. In fact,some N370S/N370S patients are asymptomatic throughout most of their lifebut may be at risk for developing neurological disorders such asParkinson's. In this case, the Gba mutation would be a risk factor forParkinson's. Additional point mutations associated with type 1 Gaucherinclude 84GG, R496H, Q350X, and H162P (Orvisky et al., Human Mutation.2002, 495, 19(4): 458-9). In addition, splice-site mutation IVS10+2T→Gand IVS10+2T→A were also associated with type I Gaucher disease(Orvisky, supra).

Neuronopathic type 2 Gaucher disease is associated with mutationsresulting primarily in two amino acid substitutions, L444P and A456P.L444P homozygosity also is commonly associated with type 3 Gaucherdisease, although this mutation has been identified in patients with allthree disease types. Other point mutations associated with types 2 and 3neuronopathic Gaucher disease include D409H (homozygotes) and V349L andD409V (heterozygotes). Patients homozygous for D409H exhibit a uniquephenotype that includes hydrocephalus and cardiac valve and aorticcalcification in addition to the neurological involvement. The lattertwo point mutations, V349L and D409V, result in Gba that iscatalytically defective. Other mutations identified in type 2 or 3disease are K198E, K198T, Y205C, F251L, 1402F, and splice-site mutationIVS10+2T→A (Orvisky et al., supra; and Lewin et al., Mol Genet Metab.2004; 81(1): 70-3). Patients and knockout mice lacking any Gba activitydie shortly after birth due to dehydration, since ceramide is essentialfor skin cutaneous integrity (Liu et al., Proc. Natl. Acad Sci. USA.1998; 95: 2503-08).

Chaperones for Gba. Isofagomine (IFG;(3R,4R,5R)-5-(hydroxymethyl)-3,4-piperidinediol) refers to a compoundhaving the following structure:

IFG has a molecular formula of C₆H₁₃NO3 and a molecular weight of147.17. This compound is further described in U.S. Pat. No. 5,844,102 toSierks et al., and U.S. Pat. No. 5,863,903, to Lundgren et al.

C-benzyl-IFG, refers to a compound having the following structure:

Other chaperones for Gba include glucoimidazole, polycyclohexanyl, andhydroxyl piperidine derivatives, which are described in pending U.S.published applications 2005/0130972 and 2005/0137223, and correspondingPCT publications WO 2005/046611 and WO 2005/046612, all filed on Nov.12, 2004 and incorporated herein by reference. Glucoimidazole andderivatives are represented by the following chemical structure:

wherein B is selected from the group consisting of hydrogen, hydroxy,acetamino, and halogen;

R¹ and R² optionally present are short, flexible linkers linear lengthof about 6 Å to about 12 Å, preferably about 9 Å. R¹ and R² can also beindependently selected from the group consisting of C₂-C₆ substituted orunsubstituted alkyl optionally interrupted by one or more moietieschosen from the group consisting of NH, NHCOO, NHCONH, NHCSO, NHCSNH,CONH, NHCO, NR³, O, S, S(O)_(m) and —S(O)_(m) NR³; C₂-C₆ substituted orunsubstituted alkenyl optionally interrupted by one or more moietieschosen from the group consisting of NH, NHCOO, NHCONH, NHCSO, NHCSNH,CONH, NHCO, NR³, O, S, S(O)_(m) and —S(O)_(m) NR³; C₂-C₆ substituted orunsubstituted alkynyl optionally interrupted by one or more moietieschosen from the group consisting of NH, NHCOO, NHCONH, NHCSO, NHCSNH,CONH, NHCO, NR³, O, S, S(O)_(m) and —S(O)_(m) NR³ , whereas m is 1 or 2,and R³ is independently selected from each occurrence from the groupsconsisting of hydrogen substituted or unsubstituted alkyl, substitutedor unsubstituted alkenyl; substituted or unsubstituted alknyl;substituted or unsubstituted cycloalkyl, substituted or unsubstitutedcycloalkenyl; substituted or unsubstituted aryl; substituted orunsubstituted arylalkyl; substituted or unsubstituted heteroaryl;substituted or unsubstituted heterocyclic; substituted or unsubstitutedheterocyclyalkyl; substituted or unsubstituted heteroarylalkyl; andpharmaceutically acceptable salts and prodrugs thereof.

In addition, R¹-L¹ or R²-L² can be a hydrogen, if either R²-L² or R¹-L¹is other than a hydrogen, respectively.

R⁵ represents a hydrogen, hydroxy, or hydroxylmethyl;

L¹ and L² are lipophilic groups selected from the group consisting ofC₃-C₁₂ substituted or unsubstituted alkyl, substituted or unsubstitutedalkenyl substituted or unsubstituted alkynyl; substituted orunsubstituted cycloalkyl, substituted or unsubstituted cycloalkenyl;substituted or unsubstituted aryl; substituted or unsubstitutedarylalkyl; substituted or unsubstituted heteroaryl; substituted orunsubstituted heterocyclic; substituted or unsubstitutedheterocycloalkyl; substituted or unsubstituted heteroarylalkyl.

In specific embodiments, GIZ compounds include (5R, 6R, 7S,8S)-5-hydroxymethyl-2-octyl-5,6,7,8-tetrahydroimidazo[1,2-a]pyridine-6,7,8-trioland (5R, 6R, 7S,8S)-5-Hydroxymethyl-2-(3,3-dimethylbutyl)-5,6,7,8-tetrahydroimidazo[1,2-a]pyridine-6,7,8-triol.

Polyhydroxylcycloalkyl (PHCA) derivatives contemplated for use in thepresent invention include compounds represented by the followingchemical structure:

wherein B is selected from the group consisting of hydrogen, hydroxy,N-acetamino, and halogen.

R¹ is independently selected for each occurrence from the groupconsisting of hydrogen; substituted or unsubstituted alkyl, substitutedor unsubstituted alkenyl, substituted or unsubstituted alkynyl,substituted or unsubstituted cycloalkyl substituted or unsubstitutedcycloalkenyl, substituted or unsubstituted aryl, substituted orunsubstituted arylalkyl, substituted or unsubstituted heteroaryl,substituted or unsubstituted heterocyclic, substituted or unsubstitutedheterocyclyalkyl, substituted or unsubstituted heteroarylalkyl, —C(O)R³and —S(O)_(m)R³, whereas m is 1 or 2, and R³ is independently selectedfor each occurrence from the groups consisting of hydrogen, substitutedor unsubstituted alkyl, substituted or unsubstituted alkenyl;substituted or unsubstituted alknyl; substituted or unsubstitutedcycloalkyl, substituted or unsubstituted cycloalkenyl; substituted orunsubstituted aryl; substituted or unsubstituted arylalkyl; substitutedor unsubstituted heteroaryl; substituted or unsubstituted heterocyclic;substituted or unsubstituted heterocyclyalkyl; substituted orunsubstituted heteroarylalkyl, and —C(O) attached to a C₁-C₆ substitutedor unsubstituted alkyl.

R² optionally present is a short, flexible linker linear length of about6 Å to about 12 Å, preferably, about 9 Å. R² can aso be selected fromthe group consisting of C₂-C₆ substituted or unsubstituted alkyloptionally interrupted by one or more moieties chosen from the groupconsisting of NH, NHCOO, NHCONH, NHCSO, NHCSNH, CONH, NHCO, NR³, O, S,S(O)_(m) and —S(O)_(m) NR³; C₂-C₆ substituted or unsubstituted alkenyloptionally interrupted by one or more moieties chosen from the groupconsisting of NH, NHCOO, NHCONH, NHCSO, NHCSNH, CONH, NHCO, NR³, O, S,S(O)_(m) and —S(O)_(m) NR³; C₂-C₆ substituted or unsubstituted alkynyloptionally interrupted by one or more moieties chosen from the groupconsisting of NH, NHCOO, NHCONH, NHCSO, NHCSNH, CONH, NHCO, NR³, O, S,S(O)_(m) and —S(O)_(m)NR³ , whereas m is 1 or 2, and R³ is independentlyselected for each occurrence from the groups consisting of hydrogen,substituted or unsubstituted alkyl, substituted or unsubstitutedalkenyl; substituted or unsubstituted alknyl; substituted orunsubstituted cycloalkyl, substituted or unsubstituted cycloalkenyl;substituted or unsubstituted aryl; substituted or unsubstitutedarylalkyl; substituted or unsubstituted heteroaryl; substituted orunsubstituted heterocyclic; substituted or unsubstitutedheterocyclyalkyl; substituted or unsubstituted heteroarylalkyl, and—C(O) attached to a C₁-C₆ substituted or unsubstituted alkyl; andpharmaceutically acceptable salts and prodrugs thereof.

L is a lipophilic group selected from the group consisting of C₃-C₁₂substituted or unsubstituted alkyl, substituted or unsubstitutedalkenyl, substituted or unsubstituted alkynyl; substituted orunsubstituted cycloalkyl; substituted or unsubstituted cycloalkenyl;substituted or unsubstituted aryl; substituted or unsubstitutedarylalkyl; substituted or unsubstituted heteroaryl; substituted orunsubstituted heterocyclic; substituted or unsubstitutedheterocyclyalkyl; substituted or unsubstituted heteroarylalkyl.

Hydroxylpiperidine derivatives contemplated for use in the presentinvention where Gba is mutated are represented by the following chemicalstructure.

wherein A represents a carbon or nitrogen;

B is a hydrogen, hydroxyl, N-acetamide or a halogen;

R¹ is a hydrogen, substituted or unsubstituted: alkyl, alkenyl, alkynyl,cycloalkyl, cycloalkenyl, aryl, arylalkyl, heteroaryl, heterocyclic,heterocyclyalkyl, or heteroarylalkyl; —C(O)R³ or —S(O)_(m)R³.Preferably, R¹ comprises H or an organic moiety having 1-12 carbonatoms.

R² is an optional short, flexible linker with a linear length of fromabout 6 Å to about 12 Å. Alternatively, R² is a C₁-C₆ substituted orunsubstituted: alkyl, alkenyl, or alkynyl optionally interrupted by oneor more moieties chosen from the group consisting of NH, NHCOO, NHCONH,NHCSO, NHCSNH, CONH, NHCO, NR³, O, S, S(O)_(m) and —S(O)_(m)NR³.

R³ is of hydrogen, or a substituted or unsubstituted: alkyl, alkenyl;alknyl; cycloalkyl, cycloalkenyl; aryl; arylalkyl; heteroaryl;heterocyclic; heterocyclyalkyl; or heteroarylalkyl. Preferably, R³comprises H or an organic moiety having 1-12 carbon atoms, or morepreferably 1-6 carbon atoms.

m is 1 or 2, and

R⁵ is a hydrogen, hydroxyl, or hydroxymethyl.

L is a lipophilic group having 1-12 carbon atoms comprising asubstituted or unsubstituted: alkyl, alkenyl, alkynyl; cycloalkyl,cycloalkenyl; aryl; arylalkyl; heteroaryl; heterocyclic;heterocycloalkyl; or heteroarylalkyl.

In specific embodiments, hydroxyl piperidene compounds contemplated foruse in the present invention include but are not limited to thefollowing; (3R,4R,5R,6S/6R)-5-(hydroxymethyl)-6-n-butyl-3,4-dihydroxypiperidine; (3R,4R,5R,6S/6R)-5-(hydroxymethyl)-6-n-hexyl-3,4-dihydroxypiperidine; (3R,4R,5R,6S/6R)-5-(hydroxymethyl)-6-n-heptyl-3,4-dihydroxypiperidine; (3R,4R,5R,6S/6R)-5-(hydroxymethyl)-6-n-octyl-3,4-dihydroxypiperidine; (3R,4R,5R,6S/6R)-5-(hydroxymethyl)-6-n-nonyl-3,4-dihydroxypiperidine; (3R,4R,5R,6S/6R)-5-(hydroxymethyl)-6-benzyl-3,4-dihydroxypiperidine.

Still other chaperones for Gba are described in U.S. Pat. No. 6,599,919to Fan et al., and include calystegine A₃, calystegine A₅, calystegineB₁, calystegine B₂, calystegine B₃, calystegine B₄, calystegine C₁,N-methyl-calystegine B₂, DMDP, DAB, castanospemine, 1-deoxynojirimycin,N-butyl-deoxynojirimycin, 1-deoxynojirimycin bisulfite,N-butyl-isofagemine, N-(3-cyclohexylpropyl)-isofagomine, N-(3-phenylpropyl)-isofagomine, andN-[(2E,6Z,10Z)-3,7,11-trimethyldodecatrienyl]-isofagomine.K

In another specific embodiment following are specific pharmacologicalchaperones including 1-deoxynojirimycin (DNJ;1,5-imino-1,5-dideoxy-D-glucitol-CAS No. 19130-96-2) and derivativeswhich can be used for treating neurological risk factors, conditions ordisorders in which the lysosomal enzyme α-glucosidase (Gaa) is mutated.

Exemplary mutations of Gaa include the following: D645E (Lin et al.,Zhonghua Min Guo Xiao Er Ke Yi Xue Hui Za Zhi. 1996;37(2): 115-21);D645H (Lin et al., Biochem Biophys Res Commun. 1995 17;208(2): 886-93);R224W, S619R, and R660H (New et al. Pediatr Neurol. 2003;29(4): 284-7);T1064C and C2104T (Montalvo et al., Mol Genet Metab. 2004;81(3): 203-8);D645N and L901Q (Kroos et al., Neuromuscul Disord. 2004;14(6): 371-4);G219R, E262K, M408V (Fernandez-Hojas et al., Neuromuscul Disord.2002;12(2): 159-66); G309R (Kroos et al., Clin Genet. 1998;53(5):379-82); D645N, G448S, R672W, and R672Q (Huie et al., Biochem BiophysRes Commun. 1998; 27;244(3): 921-7); P545L (Hermans et al., Hum MolGenet. 1994;3(12): 2213-8); C647W (Huie et al., Hum Mol Genet.1994;3(7): 1081-7); G643R (Hermans et al., Hum Mutat. 1993;2(4):268-73); M318T (Zhong et al., Am J Hum Genet. 1991;49(3): 635-45); E521K(Hermans et al., Biochem Biop hys Res Commun. 1991;179(2): 919-26);W481R (Raben et al., Hum Mutat. 1999;13(1): 83-4); and L552P and G549R(unpublished data).

Splicing mutants include IVSIAS, T>G, −13 and IVS8+1G>A).

Exemplary α-glucosidase chaperones are represented by the followingchemical structure:

-   wherein:-   R₁ is H or a straight or branched alkyl, cycloalkyl, alkenyl,    alkylether or alkyl amine containing 1-12 carbon atoms, an aryl,    alkylaryl, heteroaryl, or heteroaryl alkyl containing 5-12 ring    atoms, where R₁ is optionally substituted with one or more —OH,    —COOH, —Cl, —F, —CF₃, —OCF₃, —O—C(═O)N-(alkyl)₂; and-   R₂ is H; a straight or branched alkyl, cycloalkyl, alkenyl, or    alkylether, containing 1-9 carbon atoms or aryl containing 5-12    carbon atoms, wherein R₂ is optionally substituted with —OH, —COOH,    —CF₃, —OCF₃ or a heterocyclic ring;-   wherein at least one of R₁ and R₂ is not H, or a pharmaceutically    acceptable salt thereof.

In particular, chaperones for acid a-glucosidase include but are notlimited to N-methyl-DNJ, N-ethyl-DNJ, N-propyl-DNJ, N-butyl-DNJ,N-pentyl-DNJ, N-hexyl-DNJ, N-heptyl-DNJ, N-octyl-DNJ, N-nonyl-DNJ,N-methylcyclopropyl-DNJ, and. N-methylcyclopentyl-DNJ.

In addition to the nitrogen-substituted DNJ derivatives, other DNJderivatives useful as chaperones for Gaa include N-benzyl substitutedDNJ derivatives and derivatives having a substituent appended to the C-1carbon adjacent to the ring nitrogen are also preferred compounds of thepresent invention. Such compounds are described in commonly-ownedco-pending application Ser. No. 11/440,473, filed on May 17, 2006.

In yet another embodiment, preferred chaperones for treatment ofneurological disorders associated with heterozygous mutations inα-galactosidase (α-Gal A), another lysosornal enzyme, are represented bythe following chemical structures:

-   wherein R₁ and R_(1′) represent H, OH, or a 1-12 carbon alkyl,    hydroxyalkyl or an alkoxyl group;-   R₂ and R₂ independently represent H, LH, or N-acetamido group, or a    1-12 carbon alkyl group;-   R₄ and R_(4′) independently represent H, OH;-   R₆ and R_(6′) independently represent H, CH2OH, CH3, or COOH;-   R₇ represents H or OH;-   R₀ represents H, methyl, or a straight chain or branched saturated    or unsaturated carbon chain containing 9-12 carbon atoms, optionally    substituted with a phenyl, hydroxyl or cyclohexyl group.

In a specific embodiment, the chaperone is 1-deoxynojirimycin.

Exemplary α-Gal A mutations associated with Fabry disease include R301Q,L166V, A156V, G272S, and M2961.

Assays

Detection and trafficking of accumulated proteins. Protein accumulationin the ER can be detected and/or visualized and manifests as perinuclearlocalization in tubulovesicular profiles that co-localize with ERresident proteins such as BiP. These proteins are also reduced or absentat their native location within the cell such as at the cell surface orin another cellular compartment such as the lysosome. Proteinaccumulation in the cytoplasm can be detected using similarco-localization methods with cytosolic proteins.

Methods for detecting impaired trafficking of lysosomal enzymes are wellknown in the art. For example, for proteins which are N- andO-glycosylated in the Golgi apparatus, pulse-chase metabolic labelingusing radioactively labeled proteins, combined with glycosidasetreatment and immunoprecipitation, can be used to detect whether theproteins are undergoing full glycosylation in the Golgi, or whether theyare being retained in the ER instead of trafficking to the Golgi forfurther glycosylation.

Sensitive methods for visually detecting cellular localization ofproteins also include fluorescent microscopy using fluorescent proteinsor fluorescent antibodies. For evaluation of cell samples, fluorescentantibodies can be used to detect proteins. For detection in manipulatedor engineered cells, proteins of interest can be tagged with e.g., greenfluorescent protein (GFP), cyan fluorescent protein, yellow fluorescentprotein (YFP), and red fluorescent protein, prior to transfection,followed by multicolor and time-lapse microscopy and electron microscopyto study the fate of the proteins in fixed cells and in living cells.For a review of the use of fluorescent imaging in protein trafficking,see Watson et al., Adv Drug Deliv Rev. 2005; 57(1): 43-61). For adescription of the use of confocal microscopy for intracellularco-localization of proteins, see Miyashita et al., Methods Mol Biol.2004; 261: 399-410.

In addition, dual labeling experiments with antibodies to, e,g, LAMP-1or LysoTracker® for the lysosome (red) (or another stain or markerspecific for the lysosome such as fluorescent quantum dots, Cascade bluedextran), and lysosomal enzyme (green), green/red overlap ratios(co-localization) can be used to measure changes in lysosomal enzyme,e.g., enzyme trafficking to the lysosomes (increasing green/red ratiomeans more enzyme is trafficked to the lysosome). Normal healthy cellswith normal endocytic pathways should yield more fluorescence. See alsoExample 2, infra.

Fluorescence correlation spectroscopy (FCS) is an ultrasensitive andnon-invasive detection method capable of single-molecule and real-timeresolution (Vukojevic et al., Cell Mol Life Sci. 2005; 62(5): 535-50).SPFI (single-particle fluorescence imaging) uses the high sensitivity offluorescence to visualize individual molecules that have beenselectively labeled with small fluorescent particles (Cherry et al.,Biochem Soc Trans. 2003; 31(Pt 5): 1028-31), For localization ofproteins within lipid rafts, see Latif et al., Endocrinology. 2003;144(11): 4725-8). For a review of live cell imaging, see Hariguchi, CellStruct Funct. 2002; 27(5): 333-4).

Fluorescence resonance energy transfer (FRET) microscopy is also used tostudy the structure and localization of proteins under physiologicalconditions (Periasamy, J Biomed Opt. 2001; 6(3): 287-91).

In particular embodiments, detection of a-synuclein in individualsharboring Gba mutations can be done using ELISA or western-blotanalysis. LCMS/MS methods and/or TLC can be used to monitor GluCerlevels (substrate accumulation).

Ex vivo monitoring of a-synuclein levels and oligomer/monomer ratios, inresponse to treatment of animals with inhibitors and/or chaperones, canbe assessed using brain slice assays.

Ubiquitination assays. In addition, assays to determine the presence andlocalization of ubiquitin-lysosomal enzyme conjugates can be used toassess toxic gain of function effects of mutations and in response tochaperone treatment. Morphological studies using immunohistochemistry orimmunofluorescence to localize these conjugates is one sensitive methodof detection. See Example 3, infra. As indicated above, the presence oflow levels of ubiquitinated proteins compared with non-stressed cellscan be indicative of inhibition of proteasome function.

As another example, a process called AlphaScreen™ (Perkin-Elmer) can beused to detect ubiquitinated proteins. In this model, the OST moiety ofa GST-UbcH5a fusion protein is ubiquitinated using biotin-Ubiquitin(bio-Ub). Following ubiquitin activation by E1, in the presence of ATP,bio-Ub is transferred to UbcH5a. In this reaction, UbcH5a acts as thecarrier to transfer the bio-Ub to its tagged GST moiety. The proteinwhich becomes biotinylated and ubiquitinated is then captured byanti-GST Acceptor and streptavidin. Donor beads resulting in signalgeneration. No signal is be generated in the absence of ubiquitination.

In addition, high throughput assays for measuring the activities of thevarious E3 ubiquitin ligases and E2 conjugation enzymes can be used todetermine the increase or decrease in protein ubiquitination (Meso ScaleDiscovery, Gaithersburg, Md.).

UPR response. ER stress, can be evaluated by determining the expressionlevels of genes and the proteins encoded by the genes involved in theUPR. Such genes and proteins include those mentioned above, Grp78/BiP,Grp94, and orp150, which are upregulated in the early stages of the UPR.Other proteins involved in the ER stress response include IRE1, PERK,ATF6, and XBP1, which are up-regulated in cells subjected to continuedER stress. Further, prolonged cell stress leads to apoptosis, and thus,upregulation of jun kinase (JNK) and caspases 3, 9 and 12.

The present invention contemplates comparison of expression levels ofthe aforementioned indicator genes and/or proteins among patients withtoxic protein or substrate accumulation or aggregation and healthyindividuals.

In another embodiment, the present invention also contemplatesevaluating the effect of specific pharmacological chaperones on stressedcells to identify compounds for relieving the cell stress caused bytoxic gain of function aggregates. As positive controls, ER stressinducers such as tunicamycin, dithiiothreitol (DTT), lacatcystin, andperoxide can be used to cause accumulation of unfolded proteins in theER. Tunicamycin inhibits N-linked glycosylation and DTT preventsdisulfide bond formation. Lacatcyctin is a proteasome inhibitor. Stressrelievers such as cyclohexamide, a protein synthesis inhibitor, can beused as positive controls when evaluating chaperone compounds onstressed cells.

Assays for expression levels include gene expression via microarrayanalysis. This can be achieved using e.g., Affyrnetrix U133 gene chipset (human genome) contain such genes (Affymetrix, Santa Clara, Calif.).In addition, this technique has been used by others. For example,microarray analysis of RNA collected from multiple time points following6-hydroxydopamine (6-OHDA) treatment was combined with data mining andclustering techniques to identify distinct functional subgroups of cellstress genes (Holtz et al., Antioxidants & Redox Signaling. 2005; 7:639-648). 6-OHDA is a parkinsonian mimetic has been shown to causetranscriptional changes associated with cellular stress and the UPR.

Apoptosis. In addition, as stated above, prolonged, persistent ER stressthat is not eliminated by the UPR can also lead to programmed cell deathin neuronal cells, e.g., apoptosis. For in vitro evaluation, neuronalcell lines such as hNT2 (ATCC accession # CRL-10742), Hs68 (# CRL-1636),HCN-1A (# CRL-10442), SK-N-F1 (# CRL-2142), SK-N-DZ (# CRL-2149),SK-N-SH (# HTB-11), or NT2/D1 (# CRL-1973), or embryonic stem cells orneural stem cells that have been differentiated in vitro to neurons(see, e.g., US 2003/0013192 to Laeng et al.; and Yan et al., Stem Cells.2005; 23: 781-90), can be transfected with mutant Gba and evaluated forapoptosis.

Thus, the number of apoptotic cells can be measured using fluorescentsubstrate analogs for, e.g., caspase 3, an early indicator of apoptosis.Apoptosis can be detected using numerous methods in the art, includingfluorescent activated cell sorting (FACS), and/or using a fluorescentplate reader (e.g., 96 wells for high-throughput). For the latter, thepercentage of cells positive for apoptosis or cell death can bedetermined, or fluorescence intensity can be measured relative to theprotein concentration.

Cell/organelle morphology. Morphological abnormalities in neurons canresult from mutant protein accumulation and can be evaluated usingmorphometric analysis. For example, changes in neuron morphology inneurons transfected with tau-GFP included asymmetry, a reduction in thenumber of axons in the anterior and posterior projections, abnormal axonbundling, axon Webbing, and reduced terminal arborisations. Otheralterations in cell morphology including aggregation, cell size (cellarea or cell density), polymegathism (variation of cell size such ascoefficient of variation of mean cell area), pleomorphism (variation ofcell shape such as percent of hexagonal cells or coefficient ofvariation of cell shape), cell perimeter, average cell side length, cellshape, and so forth. Morphology can be evaluated using quantitativemorphometric analysis according to methods described in, Ventimiglia etal., J Neurosci Methods. 1995; 57: 63-6; and Wu et al., Cerebral Cortex.2004; 14: 543-54 (high-throughput analysis); and using image analysissoftware such as Image Pro-Plus software

Cell/ER stress can also be detected by evaluating organelle morphology.For example, the UPR in CY028-expressing S. cerevisiae cells wasmanifested as an aberrant morphology of the endoplasmic reticulum (ER)and as extensive membrane proliferation compared to the ER morphologyand membrane proliferation of wild-type CY000-producing S. cerevisiaecells (Sagt et al., Applied and Environmental Microbiology. 2002; 68:2155-2160).

Moreover, specific morphological indicators can be associated withindividual aggregation diseases. For example, in Gaucher disease, thelipids accumulate in lysosomes of macrophages resulting in a distinctmorphology indicative of an activated macrophage.

ER calcium stores. ER stress also can be detected by measuring thelevels of calcium in the ER lumen and cytosol, and also by determiningthe level of calcium regulatory proteins such as SERCA2b, a ubiquitouscalcium-ATPase which regulates intracellular calcium stores. As acontrol, ER stress can be induced by calcium depletion, using, e.g.,thapsigargin.

Proteasome function. Proteasome function, one cell stress response toaccumulation of proteins or substrates, can be measured according to themethod of Glas et al. (Nature. 1998; 392: 618-622). Evaluation of 26Sproteasome function in living animals by imaging has been achievedubiquitin-luciferase reporter for bioluminescence imaging (Luker et al.,Nature Medicine. 2003. 9, 969-973). Proteasome isolation and assays aredescribed in Craiu et al., JBC. Kits for proteasome isolation arecommercially available from, for example, Calbiochem (Cat. No. 539176).This kit can be used to isolate proteasome subunits from cell extractsto study their function and interactions with other proteins. Theproteasome subunits can be identified by loading the beads directly ontoan SDS-PAGE gel and immunoblotting with subunit specific antibodies.Alternatively, proteasome bound to the beads can be used in proteolyticassays using proteasome substrates.

pH cell growth and trafficking assays. Trafficking of proteins in cellsoccurs along pH gradients (i.e., ER pH about 7.0, Golgi pH about6.2-7.0, trans-Golgi network pH about 6.0, early and late endosomes pHabout6.5, lysosomes pH about 4.5). Trafficking, lysosome/endosomemorphologies, and luminal pHs are also disrupted in some lysosomalstorage diseases (Ivleva et al., Biomed Sci. 1991; 2: 398-402; Futermanand van Meer, Nat Rev Mol Cell Biol, 2004; 5: 554-65), and elevated pHin the endosome has been shown to promote a reversal of vesiculartrafficking from endosomes to Golgi (van Wert et al., 1995, supra).

The growth rate of cells (e.g., wild- type, untreated patient cells andchaperone treated patient cells) exposed to a range of pHs can bemeasured and compared using a fluorescent plate reader. Apoptosis andcell death assays (described above) can also be used to determinepH-sensitivity on cell viability.

Alternatively, lysosomal pH and pH effects on trafficking can beevaluated using a confocal microscope. pH-sensitive fluorescent probesthat are endocytosed by the cells can be used to measure pH ranges inthe lysosomes and endosomes (i.e., fluorescein is red at pH 5.0 and blueto green at pH 5.5 to 6.5). Lysosome morphology and pH can be comparedin wild type and chaperone treated and untreated patient cells. Thisassay can be run in parallel with the plate reader assay to determinethe pH-sensitivity. In addition, trafficking of enzymes to the lysosomecan be evaluated in cells at different pH's using the dual labelingexperiments described above.

Rates of endocytosis for cells (wild type, chaperone treated anduntreated patient cells) exposed to various pHs can be measured usingQuantum dots or Dextran Blue. In addition, assays describing the use offluorescent lipid analogs (BODIPY-LacCer, -GM1 gangliosides etc.) aredescribed in Pagano, Phil Trans R Soc Lond B. 2003; 358-885-91.

Enzyme activity. In addition to evaluating the effect of chaperones onaggregation and/or trafficking, using the protein localization assaysdescribed above, biochemical assays can also be used to determinewhether the proteins are functional, and to assess the effects ofrestoring function, once they have been chaperoned out of the ER, e.g.,to the lysosome. Activity assays are generally designed to measure theactivity of a target protein in the presence or absence of a test agent.Such assays will depend on the specific protein. For example, where theprotein is an enzyme, intracellular enzyme activity assays usingsubstrates are routine in the art can be used to assess enzyme activity.

Ex vivo and in vivo evaluation of enzyme activity can be performed usingnormal animals and animal models of disease states such as describedinfra.

Methods of Diagnosis

The present invention provides a method for diagnosing a risk factor,condition, or neurological disorder associated with a mutation in alysosomal enzyme. Since neurological effects which occur in patientswith LSDs can be present in other neurological disorders, persons withmutations in the lysosomal enzymes, but who have not been diagnosed withan LSD may not be effectively treated. One example is individuals withheterozygous mutations in the Gba gene, who are at risk of developing,or have developed parkinsonism or Parkinson's disease. Other exemplaryneurological symptoms that may be associated with a mutant lysosomalenzyme include neurodegeneration, neurological regression, seizures,blindness, eye movement disorders, spacisticity, dementia; developmentaldelays; neuromuscular symptoms, peripheral neuropathy (neuropathicpain), acroparesthesia, impairments in long-term memory, cerebrovascularevents such as cerebrovascular events (stroke, transient ischemicattack), and impaired swallowing.

Methods of identifying a mutation or mutations in lysosomal enzymes arewell-known in the art and include comparing enzyme activity of alysosomal enzyme from a biological sample from an individual exhibitingneurological symptoms, or an individual who is at risk of developingneurological symptoms (such as a carrier for an LSD or a relative of anindividual having a LSD. Methods of identifying mutations at a molecularlevel, i.e., nucleotide or amino acid alterations, also are well knownto those skilled in the art, such as PCR amplification followed bysequencing, single strand conformation polymorphism (SSCP) or using DNAmicroarrays for large samples (Tennis et al., Cancer EpidemiologyBiomarkers & Preventio, 2006:15: 80-85)

Formulation, Dosage, and Administration of Specific PharmacologicalChaperones

The present invention provides that the specific pharmacologicalchaperone be administered in a dosage form that permits systemicadministration, since the compounds need to cross the blood-brainbarrier to exert effects on neuronal cells. In one embodiment, thespecific pharmacological chaperone is administered as monotherapy,preferably in an oral dosage form (described further below), althoughother dosage forms are contemplated. In one embodiment, it iscontemplated that the dosing regimen should be one that provides aperiodic peak level of compound in the plasma of the individual beingtreated. Other embodiment may require constant, steady state levels ofcompound in plasma. This can be obtained either by daily administrationin divided doses, or controlled-release formulations, or by lessfrequent administration of sustained-release dosage forms. Formulations,dosage, and routes of administration for the specific pharmacologicalchaperone are detailed below.

Formulations

The specific pharmacological chaperone can be administered in a formsuitable for any route of administration, including e.g., orally in theform tablets or capsules or liquid, or in sterile aqueous solution forinjection. When the specific pharmacological chaperone is formulated fororal administration, the tablets or capsules can be prepared byconventional means with pharmaceutically acceptable excipients such asbinding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidoneor hydroxypropyl methylcellulose); fillers (e.g., lactose,microcrystalline cellulose or calcium hydrogen phosphate); lubricants(e.g., magnesium stearate, talc or silica); disintegrants (e.g., potatostarch or sodium starch glycolate); or wetting agents (e.g., sodiumlauryl sulphate). The tablets may be coated by methods well known in theart. Liquid preparations for oral administration may take the form of,for example, solutions, syrups or suspensions, or they may be presentedas a dry product for constitution with water or another suitable vehiclebefore use. Such liquid preparations may be prepared by conventionalmeans with pharmaceutically acceptable additives such as suspendingagents (e.g., sorbitol syrup, cellulose derivatives or hydrogenatededible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueousvehicles (e.g., almond oil, oily esters, ethyl alcohol or fractionatedvegetable oils); and preservatives (e.g., methyl orpropyl-p-hydroxybenzoates or sorbic acid). The preparations may alsocontain buffer salts, flavoring, coloring and sweetening agents asappropriate. Preparations for oral administration may be suitablyformulated to give controlled or sustained release of the specificpharmacological chaperone.

The pharmaceutical formulations of the specific pharmacologicalchaperone suitable for parenteral injectable use generally includesterile aqueous solutions (where water soluble), or dispersions andsterile powders for the extemporaneous preparation of sterile injectablesolutions or dispersion. In all cases, the form must be sterile and mustbe fluid to the extent that easy syringability exists. It must be stableunder the conditions of manufacture and storage and must be preservedagainst the contaminating action of microorganisms such as bacteria andfungi. The carrier can be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and polyethylene glycol, and the like), suitable mixturesthereof, and vegetable oils. The proper fluidity can be maintained, forexample, by the use of a coating such as lecithin, by the maintenance ofthe required particle size in the case of dispersion and by the use ofsurfactants. Prevention of the action of microorganisms can be broughtabout by various antibacterial and antifungal agents, for example,parabens, chlorobutanol, phenol. benzyl alchohol, sorbic acid, and thelike. In many cases, it will be reasonable to include isotonic agents,for example, sugars or sodium chloride. Prolonged absorption of theinjectable compositions can be brought about by the use in thecompositions of agents delaying absorption, for example, aluminummonosterate and gelatin.

Sterile injectable solutions are prepared by incorporating the specificpharmacological chaperone in the required amount in the appropriatesolvent with various of the other ingredients enumerated above, asrequired, followed by filter or terminal sterilization. Generally,dispersions are prepared by incorporating the various sterilized activeingredients into a sterile vehicle which contains the basic dispersionmedium and the required other ingredients from those enumerated above.In the case of sterile powders for the preparation of sterile injectablesolutions, the preferred methods of preparation are vacuum drying andthe freeze-drying technique which yield a powder of the activeingredient plus any additional desired ingredient from previouslysterile-filtered solution thereof.

The formulation can contain an excipient. Pharmaceutically acceptableexcipients which may be included in the formulation are buffers such ascitrate buffer, phosphate buffer, acetate buffer, and bicarbonatebuffer, amino acids, urea, alcohols, ascorbic acid, phospholipids;proteins, such as serum albumin, collagen, and gelatin; salts such asEDTA or EGTA. and sodium chloride; liposomes; polyvinylpyrollidone;sugars, such as dextran, mannitol, sorbitol, and glycerol; propyleneglycol and polyethylene glycol (e.g., PEG-4000, PEG-6000); glycerol;glycine or other amino acids; and lipids. Buffer systems for use withthe formulations include citrate; acetate; bicarbonate; and phosphatebuffers. Phosphate buffer is a preferred embodiment.

The formulation can also contain a non-ionic detergent. Preferrednon-ionic detergents include Polysorbate 20, Polysorbate 80, TritonX-100, Triton X-114, Nonidet P-40, Octyl α-glucoside, Octyl β-glucoside,Brij 35, Pluronic, and Tween 20.

Administration

The route of administration of the specific pharmacological chaperonemay be oral (preferably) or parenteral, including intravenous,subcutaneous, intra-arterial, intraperitoneal, ophthalmic,intramuscular, buccal, rectal, vaginal, intraorbital, intracerebral,intradermal, intracranial, intraspinal, intraventricular, intrathecal,intracisternal, intracapsular, intrapulmonary, intranasal, transmucosal,transdermal, or via inhalation.

Administration of the above-described parenteral formulations of thespecific pharmacological chaperone may be by periodic injections of abolus of the preparation, or may be administered by intravenous orintraperitoneal administration from a reservoir which is external (e.g.,an i.v. bag) or internal (e.g., a bioerodable implant). See, e.g., U.S.Pat. Nos. 4,407,957 and 5,798,113, each incorporated herein byreference. Intrapulmonary delivery methods and apparatus are described,for example, in U.S. Pat. Nos. 5,654,007, 5,780,014, and 5,814,607, eachincorporated herein by reference. Other useful parenteral deliverysystems include ethylene-vinyl acetate copolymer particles, osmoticpumps, implantable infusion systems, pump delivery, encapsulated celldelivery, liposomal delivery, needle-delivered injection, needle-lessinjection, nebulizer, aeorosolizer, electroporation, and transdermalpatch. Needle-less injector devices are described in U.S. Pat. Nos.5,879,327; 5,520,639; 5,846,233 and 5,704,911, the specifications ofwhich are herein incorporated by reference. Any of the formulationsdescribed above can be administered using these methods.

Subcutaneous injections have the advantages allowingself-administration, while also resulting in a prolonged plasmahalf-life as compared to intravenous administration. Furthermore, avariety of devices designed for patient convenience, such as refillableinjection pens and needle-less injection devices, may be used with theform ulations of the present invention as discussed herein.

Dosage

The amount of specific pharmacological chaperone effective to rescue theendogenous mutant Gba can be determined on a case-by-case basis by thoseskilled in the art. Pharmacokinetics and pharmacodynamics such ashalf-life (t_(1/2)), peak plasma concentration (C_(max)), time to peakplasma concentration (t_(max)), exposure as measured by area under thecurve (AUC), and tissue distribution for both the replacement proteinand the specific pharmacological chaperone, as well as data for specificpharmacological chaperone/Gba binding (affinity constants, associationand dissociation constants, and valency), can be obtained using ordinarymethods known in the art to determine compatible amounts required tostabilize the replacement protein, without inhibiting its activity, andthus confer a therapeutic effect.

Data obtained from cell culture assay or animal studies may be used toformulate a therapeutic dosage range for use in humans and non-humananimals. The dosage of compounds used in therapeutic methods of thepresent invention preferably lie within a range of circulatingconcentrations that includes the ED₅₀ concentration (effective for 50%of the tested population) but with little or no toxicity. The particulardosage used in any treatment may vary within this range, depending uponfactors such as the particular dosage form employed, the route ofadministration utilized, the conditions of the individual (e,g.,patient), and so forth.

A therapeutically effective dose may be initially estimated from cellculture assays and formulated in animal models to achieve a circulatingconcentration range that includes the IC₅₀. The IC₅₀ concentration of acompound is the concentration that achieves a half-maximal inhibition ofsymptoms (e.g., as determined from the cell culture assays). Appropriatedosages for use in a particular individual, for example in humanpatients, may then be more accurately determined using such information.

Measures of compounds in plasma may be routinely measured in anindividual such as a patient by techniques such as high performanceliquid chromatography (HPLC) or gas chromatography.

Toxicity and therapeutic efficacy of the composition can be determinedby standard pharmaceutical procedures, for example in cell cultureassays or using experimental animals to determine the LD₅₀ and the ED₅₀.The parameters LD₅₀ and ED₅₀ are well known in the art, and refer to thedoses of a compound that is lethal to 50% of a population andtherapeutically effective in 50% of a population, respectively. The doseratio between toxic and therapeutic effects is referred to as thetherapeutic index and may be expressed as the ratio: LD₅₀/ED₅₀. Specificpharmacological chaperones that exhibit large therapeutic indices arepreferred.

The optimal concentrations of the specific pharmacological chaperone aredetermined according to the amount required to stabilize and induce aproper conformation of the recombinant protein, e.g., Gba, in viva, intissue or circulation, without persistently preventing its activity,bioavailability of the specific pharmacological chaperone in tissue orin circulation, and metabolism of the specific pharmacological chaperonein tissue or in circulation. For example, where the specificpharmacological chaperone is an enzyme inhibitor, the concentration ofthe inhibitor can be determined by calculating the IC₅₀ value of thespecific chaperone for the enzyme. Taking into considerationbioavailability and metabolism of the compound, concentrations aroundthe IC₅₀ value or slightly over the IC₅₀ value can then be evaluatedbased on effects on enzyme activity, e.g., the amount of inhibitorneeded to increase the amount of enzyme activity or prolong enzymeactivity of the administered enzyme. As an example, the IC₅₀ value ofthe compound isofagomine for the Gba enzyme is 0.04 μM, indicating thatit is a potent inhibitor.

Combination Drug Therapy

The specific pharmacological chaperone can be used to treat patientswith CNS disorders that are associated with mutations in lysosomalenzymes in combination with other drugs that are also used to treat theCNS disorder.

For example, for patients having Parkinson's disease, such as dopaminereceptor agonists, anticholinergics, COMT inhibitors, monoamine oxidaseB inhibitors. Exemplary agents include but are not limited to levodopa(Sinemet®; Merck), Parlodel® (bromocriptine mesylate; Novartis); Permax®(pergolide mesylate; Eli Lilly); Requip® (ropinirole HCl), Mirapex®(pramipexole dihydrochloride); Cogetin® (benztropine mesylate); Artane®(trihexyphenidyl HCl; American Cyanamid); Symmetrel® amantadinehydrochloride; Du Pont Merck); and Eldepryl® (Somerset Pharmaceuticals).

Combination Therapy with Gene Therapy

Although not yet approved for therapeutic treatment in the UnitedStates, gene therapies (both ex viva and direct transfer) for numerousgenetic disorders are under investigation. The present invention alsocontemplates use of the specific pharmacological chaperone incombination with gene therapy to replace the defective Gba gene in theneurological disease. Such a combination will enhance the efficacy ofgene therapy by increasing the level of expression of the therapeuticGba in vivo, since, in addition to enhancing folding and processing ofmutated enzymes, specific pharmacological chaperones have been shown toenhance folding and processing of the wild-type or conformationallystable counterparts (see, e.g., U.S. Pat. No. 6,274,597 to Fan et al.,Example 3).

U.S. Pat. No. 6,309,634 to Bankiewicz describes a gene therapy approachfor treating Parkinson's disease. According to the method, recombinantadeno-associated virus (rAAV) virions are produced in vitro and comprisea nucleic acid sequence encoding aromatic amino acid decarboxylase(AADC). Another group recently inserted the gene for glial cellline-derived neurotrophic factor (GDNF), also via recombinantadeno-associated viral vectors, in a monkey model of Parkinson's disease(Eslamboli et al., J Neurosci. 2005; 25(4):76977).

Any of the methods for gene therapy which are or become available in theart can be used to deliver therapeutic genes. Exemplary methods aredescribed below. For general reviews of the methods of gene therapy, seeGoldspiel et al., Clinical Pharmacy 1993, 12:488-505; Wu and Wu,Biotherapy 1991, 3: 87-95; Tolstoshev, Ann. Rev. Pharmacol. Toxical.1993, 32: 573-596; Mulligan, Science. 1993, 260: 926-932; and Morgan andAnderson, Ann. Rev. Biochem. 1993, 62: 191-217; May, TIBTECH 1993, 11:155-215, Methods commonly known in the art of recombinant DNA technologythat can be used are described in Ausubel et al., (eds.), 1993, CurrentProtocols in Molecular Biology, John Wiley & Sons, NY; Kriegler, 1990,Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY;and in Chapters 12 and 13, Dracopoli et al., (eds.), 1994, CurrentProtocols in Human Genetics, John Wiley & Sons, NY; and Colosimo et al.,Biotechniques 2000;29(2): 314-8, 320-2, 324.

The gene to be administered for the methods of the present invention canbe isolated and purified using ordinary molecular biology, microbiology,and recombinant DNA techniques within the skill of the art. For example,nucleic acids encoding the target protein can be isolated usingrecombinant DNA expression as described in the literature. See, e.g.,Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual,Second Edition (1989) Cold Spring Harbor Laboratory Press, Cold SpringHarbor, New York; DNA Cloning: A Practical Approach, Volumes I and 11(D. N. Glover ed. 1985); Oligonucleotide Synthesis (M. J. Gait ed.1984); Nucleic Acid Hybridization [B. D. Hames & S. J. Ê Higgins eds.(1985)]; Transcription And Translation [B. D. Hames & S. J. Higgins,eds. (1984)]; Animal Cell Culture [R. I. Freshney, ed. (1986)];Immobilized Cells And Enzymes [IRL Press, (1986)]; B. Ê Perbal, APractical Guide To Molecular Cloning (1984). The nucleic acid encodingthe protein may be full-length or truncated, so long as the gene encodesa biologically active protein.

The identified and isolated Gba gene can then be inserted into anappropriate cloning vector. Vectors suitable for gene therapy includeviruses, bacteriophages, cosmids, plasmids, fungal vectors and otherrecombination vehicles typically used in the art which have beendescribed for expression in a variety of eukaryotic and prokaryotichosts, and may be used for gene therapy as well as for simple proteinexpression.

In a specific embodiment, the vector is a viral vector. Viral vectors,especially adenoviral vectors can be complexed with a cationicamphiphile, such as a cationic lipid, polyL-lysine (PLL), anddiethylaminoethyldextran (DELAE-dextran), which provide increasedefficiency of viral infection of target cells (See, e.g., PCT/US97/21496filed Nov. 20, 1997, incorporated herein by reference). Viral vectorsfor use in the present invention include vectors derived from vaccinia,herpesvirus, AAV and retroviruses. In particular, herpesviruses,especially herpes simplex virus (HSV), such as those disclosed in U.S.Pat. No. 5,672,344, the disclosure of which is incorporated herein byreference, are particularly useful for delivery of a transgene to aneuronal cell. AAV vectors, such as those disclosed in U.S. Pat. Nos.5,139,941, 5,252,479 and 5,753,500 and PCT publication WO 97/09441, thedisclosures of which are incorporated herein, are also useful sincethese vectors integrate into host chromosomes, with a minimal need forrepeat administration of vector. For a review of viral vectors in genetherapy, see McConnell et al., Hum Gene Ther. 2004; 15(11): 1022-33;Mccarty et al., Annu Rev Genet. 2004; 38: 819-45; Mah et al., ClinPharmacokinet. 2002; 41(12): 901-11; Scott et al., Neuromuscul. Disord.2002;12 Suppl 1:S23-9. In addition, see U.S. Pat. No. 5,670,488.

The coding sequences of the gene to be delivered are operably linked toexpression control sequences, e.g., a promoter that directs expressionof the gene. As used herein, the phrase “operatively linked” refers tothe functional relationship of a polynucleotide/gene with regulatory andeffector sequences of nucleotides, such as promoters, enhancers,transcriptional and translational stop sites, and other signalsequences. For example, operative linkage of a nucleic acid to apromoter refers to the physical and functional relationship between thepolynucleotide and the promoter such that transcription of DNA isinitiated from the promoter by an RNA polymerase that specificallyrecognizes and binds to the promoter, and wherein the promoter directsthe transcription of RNA from the polynucleotide.

In one specific embodiment, a vector is used in which the codingsequences and any other desired sequences are flanked by regions thatpromote homologous recombination at a desired site in the genome, thusproviding for expression of the construct from a nucleic acid moleculethat has integrated into the genome (Koller and Smithies, Proc. Natl.Acad. Sci. USA. 1989, 86: 8932-8935; Zijistra et al., Nature. 1989,342:435-438; U.S. Pat. No. 6,244,113 to Zarling et al.; and U.S. Pat.No. 6,200,812 to Pati et al.).

Gene Delivery

Delivery of the vector into a patient may be either direct, in whichcase the patient is directly exposed to the vector or a deliverycomplex, or indirect, in which case, cells are first transformed withthe vector in vitro, then transplanted into the patient. These twoapproaches are known, respectively, as in vivo and ex vivo gene therapy.

Direct transfer. In a specific embodiment, the vector is directlyadministered in vivo, where it enters the cells of the organism andmediates expression of the gene. This can be accomplished by any ofnumerous methods known in the art and discussed above, e.g., byconstructing it as part of an appropriate expression vector andadministering it so that it becomes intracellular, e.g., by infectionusing a defective or attenuated retroviral or other viral vector (see,U.S. Pat. No. 4,980,286), or by direct injection of naked DNA, or by useof microparticle bombardment (e.g., a gene gun; Biolistic, Dupont); orcoating with lipids or cell-surface receptors or transfecting agents,encapsulation in biopolymers (e.g., poly-β-1-64-N-acetylglucosaminepolysaccharide; see U.S. Pat. No. 5,635,493), encapsulation inliposomes, microparticles, or microcapsules; by administering it inlinkage to a peptide or other ligand known to enter the nucleus; or byadministering it in linkage to a ligand subject to receptor-mediatedendocytosis (see, e.g., Wu and Wu, J. Biol. Chem. 1987, 62: 4429-4432),etc. In another embodiment, a nucleic acid-ligand complex can be formedin which the ligand comprises a fusogenic viral peptide to disruptendosomes, allowing the nucleic acid to avoid lysosomal degradation, orcationic 12-mer peptides, e.g., derived from antennapedia, that can beused to transfer therapeutic DNA into cells (Mi et al., Mol. Therapy.2000, 2: 339-47). In yet another embodiment, the nucleic acid can betargeted in viva for cell specific uptake and expression, by targeting aspecific receptor (see, e.g., PCT Publication Nos. WO 92/06180, WO92/22635, WO 92/20316 arid WO 93/14188). Recently, a technique referredto as magnetofection has been used to deliver vectors to mammals. Thistechnique associates the vectors with superparamagnetic nanoparticlesfor delivery under the influence of magnetic fields. This applicationreduces the delivery time and enhances vector efficacy (Scherer et al.,Gene Therapy. 2002; 9: 102-9). Additional targeting and deliverymethodologies are contemplated in the description of the vectors, below.

In a specific embodiment, the nucleic acid can be administered using alipid carrier. Lipid carriers can be associated with naked nucleic acids(e.g., plasmid DNA) to facilitate passage through cellular membranes.Cationic, anionic, or neutral lipids can be used for this purpose.However, cationic lipids are preferred because they have been shown toassociate better with DNA which, generally, has a negative charge.Cationic lipids have also been shown to mediate intracellular deliveryof plasmid DNA (Feigner and Ringold, Nature. 1989; 337: 387).Intravenous injection of cationic lipid-plasmid complexes into mice hasbeen shown to result in expression of the DNA in lung (Brigham et al.,Am. J. Med. Sci. 1989; 298: 278). See also, Osaka et al., J. Pharm. Sci.1996; 85(6):612-618; San et al., Human Gene Therapy. 1993; 4:781-788;Senior et al., Biachemica et Biophysica Acta. 1991; 1070: 173-179);Kabanov and Kabanov, Bioconjugate Chem. 1995; 6: 7-20; Liu et al.,Pharmaceut. Res. 1996; 13; Remy et al., Bioconjugate Chem. 1994; 5:647-654; Behr, J-P., Bioconjugate Chem. 1994; 5: 382-389; Wyman et al.,Biochem. 1997; 36: 3008-3017; U.S. Pat. No. 5,939,401 to Marshall et al;and U.S. Pat. No. 6,331,524 to Scheule et al.

Representative cationic lipids include those disclosed, for example, inU.S. Pat. No. 5,283,185; and e.g., U.S. Pat. No. 5,767,099, thedisclosures of which are incorporated herein by reference, In apreferred embodiment, the cationic lipid is N4-spermine cholesterylcarbamate (GL-67) disclosed in U.S. Pat. No. 5,767,099. Additionalpreferred lipids include N4-spermidine cholestryl carbamate (GL-53) and1-(N4-spermine)-2,3-dilaurylglycerol carbamate (GL-89).

Preferably, for in vivo administration of viral vectors, an appropriateimmunosuppressive treatment is employed in conjunction with the viralvector, e.g., adenovirus vector, to avoid immuno-deactivation of theviral vector and transfected cells. For example, immunosuppressivecytokines, such as interleukin-12 (IL-12), interferon-γ (IFN-γ), oranti-CD4 antibody, can be administered to block humoral or cellularimmune responses to the viral vectors. In that regard, it isadvantageous to employ a viral vector that is engineered to express aminimal number of antigens.

Indirect transfer. Somatic cells may be engineered ex vivo with aconstruct encoding a wild-type protein using any of the methodsdescribed above, and re-implanted into an individual. This method isdescribed generally in WO 93/09222 to Selden et al. In addition, thistechnology is used in Cell Based Delivery's proprietary ImPACTtechnology, described in Payumo et al., Clin. Orthopaed. and RelatedRes. 2002; 403S: S228-S242. In such a gene therapy system, somatic cells(e.g., fibroblasts, hepatocytes, or endothelial cells) are removed fromthe patient, cultured in vitro, transfected with the gene(s) oftherapeutic interest, characterized, and reintroduced into the patient.Both primary cells (derived from an individual or tissue and engineeredprior to passaging), and secondary cells (passaged in vitro prior tointroduction in vivo) can be used, as well as immortalized cell linesknown in the art. Somatic cells useful for the methods of the presentinvention include but are not limited to somatic cells, such asfibroblasts, keratinocytes, epithelial cells, endothelial cells, glialcells, neural cells, formed elements of the blood, muscle cells, othersomatic cells that can be cultured, and somatic cell precursors. In apreferred embodiment, the cells are fibroblasts or mesenchymal stemcells.

Nucleic acid constructs, which include the exogenous gene and,optionally, nucleic acids encoding a selectable marker, along withadditional sequences necessary for expression of the exogenous gene inrecipient primary or secondary cells, are used to transfect primary orsecondary cells in which the encoded product is to be produced. Suchconstructs include but are not limited to infectious vectors, such asretroviral, herpes, adenovirus, adenovirus-associated, mumps andpoliovirus vectors, can be used for this purpose.

Mesenchymal stem cells (MSCs) are non-blood-producing stem cellsproduced in the bone marrow. MSCs can be made to differentiate andproliferate into specialized non-blood tissues. Stem cells transfectedwith retroviruses are good candidates for the therapy due to theircapacity for self-renewal. This ability precludes repetitiveadministration of the gene therapy. Another advantage is that if theinjected stem cells reach the target organ and then differentiate, theycan replace the damaged or malformed cells at the organ.

As one example, for Gaucher disease, trials are underway fortransduction of somatic stem cells from an individual with a retrovirusencoding the Gba gene, followed by returning the corrected stem cells tothe patient, where they take up residence in the bone marrow and produceCiba-expressing cells such as macrophages.

Chaperone Delivery. When administered in combination with gene therapyencoding a therapeutic gene, the specific pharmacological chaperone canbe administered according to the methods and dosage forms describedabove.

Combination with Substrate Inhibitors

In addition, combination of small molecule chaperones of this inventionwith other small molecule substrate inhibitors, as described in thebackground, is also contemplated. Since even a slight reduction inlysosomal enzyme activity can lead to elevated lipid accumulation, whichcan, in turn, alter the phospholipid balance of the cell or initiatesignaling events that result in apoptosis. Exemplary substrateinhibitors include NB-DNJ (Miglustat) for inhibition of ceramidespecific glucosyltransferases (reduction of glycolipid substrates)(Kasperzyk et al., Journal of Neurochemistry 2004. 89: 645-653).

EXAMPLES

The present invention is further described by means of the examples,presented below. The use of such examples is illustrative only and in noway limits the scope and meaning of the invention or of any exemplifiedterm. Likewise, the invention is not limited to any particular preferredembodiments described herein. Indeed, many modifications and variationsof the invention will be apparent to those skilled in the art uponreading this specification. The invention is therefore to be limitedonly by the terms of the appended claims along with the full scope ofequivalents to which the claims are entitled.

Example 1 Determination of Increased Gba Activity in the Brains of L444PTransgenic Mice Treated with Specific Pharmacological Chaperones

L444P is a mutation associated with Types 2 and 3 Gaucher disease. L444Ptransgenic mice (homozygous for human L444P mutated Gba on aglucosylceramide synthase null background) exhibit a deficiency in Gbaactivity in the brain. However, due to the disruption in theglucosylceramide synthase gene, these mice do not exhibit accumulationof GluCer in e.g., macrophages. Concomitant glucosylceramide synthasedisruption is necessary, since previously made L444P transgenic micedied within 3 days of birth due to impaired permeability barrierfunction in the epidermis.

In this experiment, the L444P transgenic mice were treated withisofagomine or C-benzyl-isofagomine and surrogate markers were measuredat 1, 3, 6 and 12 months to determine efficacy of the chaperones. Inaddition, mice in a “washout” period of 2 weeks of non-chaperonetreatment following 4 weeks of treatment were also evaluated forreversion of surrogate markers back to levels seen in untreatedcontrols.

Methods

Specific pharmacological chaperone treatment Mice were administeredisofagomine or C-benzyl-isofagomine in their drinking water, ad libitum.Estimated daily dosage based on the volume of water consumed is about 10mg/kg/day.

Gba activity assays in brain. At the end of 1, 3, 6 and 12 months, micewere sacrificed and evaluated for enhancement of Gba enzyme activity inbrain. Brian tissue is freshly harvested (blood washed away with PBS),or thawed from frozen stock. Tissue is minced tissue and homogenized onice in 200-500 μl Mcllvaine (MI) buffer (0.25% sodium taurocholate, 0.1%Triton ×-100 in 0.1M citrate and 0.2M phosphate buffer, pH 5.2), andcentrifuged at 10.000× g. The supernatant is collected and may be frozenat this step.

About 1-10 μl of supernatant from the brain tissue homogenates is addedto a clear 96-well plate for the Micro BCA Protein Assay (Pierce, cat#23235) to quantitate the amount of total protein according to themanufacturer's protocol. As a negative control, another 10 μl is addedto a black plate, mixed with 10 μl of 2.5 mM CBE (2.7 mg Conduritol BEpoxide in 6.7 ml buffer), an inhibitor of Gba activity, and left atroom temperature (RT) for 30 minutes. 50 μl of 3 mM 4-methalUmbelliferal beta-D-glucoside (4-MU-beta-D-glucoside; made fresh, powderis dissolved in 0.2 ml of DMSO, then q.s. to proper volume with MIbuffer), a Gba substrate, is then added, and the black plate is furtherincubated at 37° C. for 1 hr. After incubation, 10 μl of supernatant isadded to a second black plate, mixed with 10 μl of MI buffer and 50 μ6mM of Gba substrate 4-MU-beta-D-glucoside, and incubated at 37° C. for 1hr. The reaction is then stopped by adding 70 μl 0.2 M glycine, pH 10.8.The plate is read in a plate-reader (Victor2 1420 multilabel counter;Wallac) at F₄₆₀.

Relative beta-glucose activity is determined by the following equation:

F ₄₆₀ without CBE—F ₄₆₀ with CBE)/(A ₅₅₀ sample−A ₅₅₀ buffer)

F₄₆₀ reading is converted into nmole 4-MU based on 4-MU standard curveand A₅₅₀ is converted into mg of protein based on the protein standardcurve. One unit of Gba activity is defined as mole of 4-MU released inone hour.

Washout study. To determine if and in what time frame the effects ofdrinking water dosed AT2101 on L444P mice regress after cessation of thetreatment, a washout study was performed. Nine male 3 month old L444Pmice were dosed at about 10 mg/kg/day for 4 weeks with an equal numberof mice untreated as a control. Four treated and four untreated micewere sacrificed at the end of 4 weeks, and the remaining animals werenot further treated with isofagomine, i.e. , they were given normaldrinking water, for another two weeks prior to sacrifice and evaluationof the Gba activity in brain.

Results

Gba Activity in Brain. Significant increase in Gba activity was observedafter as little as two weeks of treatment with isofagomine in brain(FIG. 1A), which persisted through 4-12 weeks. Notably, in brain,isofagomine treatment resulted in an increase from about 1 U/mg inuntreated mice, to about 4.5 U/mg after 2 and 4 weeks of treatment, andfurther increased to about 6 U/mg after 12 weeks (p<0.001). It isexpected that increased Gba activity will persist at 3, 6 and 12 monthsand for as long as the chaperones are administered.

Similarly, after two weeks, the C-benzyl-isofagomine-treated mice alsoexhibited significant increased Gba activity in the organs such asspleen, and a trend toward increased activity in the lung and brain(data not shown). It is expected that increases in Gba activity will beobserved in other organs, including the brain, upon further treatment,since after two weeks of treatment with AT2206, there was a trend towardincrease in the brain (data not shown).

Washout. Similar to above, after 4 weeks of treatment at 10 mg/kg/day,Gba activity was significantly elevated in brain in the L444P transgenicmice. (FIG. 1B).

Discussion

These results provide the first indication that physiological levels ofchaperone are sufficient to cross the blood-brain barrier enhanceactivity of Gba in the brain and in the peripheral organs (e.g., spleenand liver). This is surprising since peripherally administered agentsoften have to be administered in higher doses to be effective in thebrain. In the case where Gba inhibitors at below-inhibitory are used aschaperones, high doses of inhibitor in the periphery would be inhibitoryfor mutant Gba, thereby defeating the purpose of enhancing enzymeactivity as previously demonstrated. Similar results were obtained inmonkeys treated with IFG, where IFG was detected in the CSF followingtreatment.

Example 2 Restoration of Disrupted Lysosomal Trafficking in GaucherFibroblasts

Although N3 70S Gaucher fibroblasts (from a human patient) do notdemonstrate an accumulation of substrate (i.e., GluCer) in thecytoplasm, these fibroblasts exhibit abnormal lysosomal protein and Gbastaining compared with wild-type fibroblasts. Treatment of N370Sfibroblasts with pharmacological chaperone isofagoniine increased theamount of Gba seen in the lysosome and restored a normal lysosomalstaining pattern to the cells.

Methods

Cell culture. N370S fibroblasts (DMN89.15) were cultured in DMEM with10% FBS and 1% penn/strep at 37C with 5% CO₂. Wild-type fibroblast cellline CRL-2097 form a healthy individual was used as a control. Cellswere sub-cultured from 10 cm plates into 12-well plates with coverslips. Cells from one confluent 10 cm plate were diluted in 38 ml ofculture medium. Isofagomine or C-benzyl-isofagomine were added from a 10mM stock solution (5% DMSO) to each well of a 12-well plate at thefollowing concentrations:

C-benzyl-isofagomine-control (secondary antibody only); untreated; 0.03μM; 0.1 μM; 0.3 μM; 1.0 μM; 3.0 μM; and 10.0 μM.

Isofagomine-control (secondary antibody only); untreated; 10 μM; 30 μM;100 μM; 1 nM; 3 nM; and 10 nM.

Cells were cultured for a total of about 6 days.

Fixing and Staining. Cells were washed for 5 minutes in PBS, fixed for15 minutes in 3.7% paraformaldahyde (in PBS), washed again for 5 minutesin PBS, and permeabilized with 0.5% saponin for 5 minutes. Cells werethen washed with PBS containing 0.1% saponin, treated for 5 minutes withfresh 0.1% sodium borohydride/0.01% saponin, and washed 3 times with PBSwith 0.1% saponin/1% BSA for 5 minutes each.

Cells were incubated for 1 h with 500 μl of primary anti-Gba (1:200) oranti-LAMP-1 (1:200; BD Pharmingen, Cat. No. 555798) antibody solution inPBS with 1% BSA. Lysosomal staining using LysoTracker® Red (Cambrex,East Rutherford, N.J.) was performed according to the manufacturer'sinstructions. Following incubation, cells were washed 3 times in 1% BSAcontaining 0.1% saponin in PBS, followed by incubation with thesecondary antibody solution (1:500; anti-rabbit AlexaFluor588 foranti-Gba and anti-mouse IgG AlexaFluor594 for anti-LAMP-1). Cells weremounted onto coverslips, sealed, and immediately viewed.

Confocal Microscopy. Cells were visualized using a confocal microscope.The red and green channel gains were set to 6 and the laser power wasoptimized using the intensity window, and were not adjusted for the restof the experiment. All slides were analyzed at the same sitting and allimages were gathered without any zoom using the 20× and 60× lens, thesmall pinhole, optimal pixel size, an average of 2 scans, and red andgreen channels were acquired simultaneously as in all previousexperiments.

All images were displayed at the same intensity and red+green channelintensity graphs were generated for each image by placing the cursorover the maximum number of cells.

Future measurements can be made by calculating a ratio for overlappingred (LAMP-1) and green (GBA) pixels.

Results

Gaucher N370S fibroblasts that have been confluent for more than 5 daysexhibit a granular lysosmal staining pattern using LysoTracker® Red(FIG. 2A) compared with a normal fibroblast, which has a punctuatestaining pattern (FIG. 2B). Similar results were shown for L444Pfibroblasts (data not shown). Staining for lysosomal LAMP-1 is shown inboth N370S and normal fibroblasts (FIGS. 2C-D, respectively). MoreLAMP-1 is shown in Gaucher fibroblasts.

Treatment with 30.0 μM isofagomine (AT2201) (FIG. 2G-H) and 3.0 μlC-benzyl-isofagomine (AT2206) (FIG. 2I-J) increased the amount of Gba inthe lysosomes and re-established a normal lysosome punctuate stainingpattern for Ciba and LAMP-1 compared with an untreated control (FIG.2E-F), as indicated by dual staining.

FIGS. 2K-N shows changes in Gba lysosomal staining in N370S Gaucherfibroblasts as follows: (K)-control (secondary antibody only);(L)-untreated N370S fibroblasts; (M)- 30 μM isofagomine; and (N) 3 μMC-benzyl-isofagomine. Gba staining is shown to localize to lysosomes inchaperone-treated versus untreated controls. Similar results wereobtained for L444P Gaucher fibroblasts (data not shown).

This improvement in normal cell morphology with chaperone treatment isdue to a decrease in the amount or accumulation of mutant Gba, possiblyin the form of aggregates, in the ER and/or cytosol. Accordingly, thisstrategy could relieve CNS symptoms in Parkinson's patients withheterozygous N370S mutations, or heterozygous Gaucher patients withhomozygous N370S mutations and parkinsonism/dementia.

Example 3 Increase of Polyubiquinated Proteins with Chaperone Treatmentin Gaucher Fibroblasts; Restoration of the Proteasome DegradationPathway

Anti-polyubiquitinated protein (PUP) and anti-Gba labeling of healthyhuman fibroblast was compared with that in fibroblasts from a Gaucherpatient having the L444P Gba mutation, and Gaucher patient fibroblastshaving the N370S Gba mutation.

Methods

Cell culture. L444P Gaucher fibroblasts (cell line GM10915); N370SGaucher fibroblasts (cell line DMN89.15); and fibroblasts from a healthyindividual (CRL-2097) were cultured in DMEM with 10% FBS and 1% PS at37C with 5% CO₂. Cells are sub-cultured from 10 cm plates into 12-wellplates with sterile cover slips. N370S cells from one confluent T-75flask were diluted 1:6 and cultured for another 4 days.

Chaperones isofagomine or C-benzyl-isofagomine are added from a 10 mMstock solution (5% DMSO) to each row of a 12-well plate at the followingconcentrations:

C-benzyl-isofagomine-untreated; control (secondary antibody only); 0.03μM; 0,1 μM; 0.3 μM; 1.0 μM; 3.0 μM; and 10.0 μM.

Isofagomine-untreated; control (secondary antibody only); 10 μM; 30 μM;100 μM; 1 nM; 3 nM; and 10 nM.

Fixing and staining. Cells are washed once in PBS for 5 minutes,followed by fixation for 15 minutes in fresh 3.7% paraformaldehyde.Cells were then washed once in PBS for 5 minutes, followed bypermeabilization for 5 minutes in 0.2% Triton X-100. Cells were thenwashed again in PBS for 5 minutes and treated for 5-10 minutes withfresh 0.1% sodium borohydride. Cells were washed three times in PBS with1% BSA (5 min each) prior to staining.

Cells are incubated for 1 hour with 500 μl of he following primaryantibodies (diluted 1:200 in PBS with 1% BSA):

-   -   1. Mouse monoclonal antibody to ubiquitinated proteins clone FK1        (AFFINITI Research Products Cat. No. PW 8805)

2. Rabbit anti-Gba antibodies are commercially available, e.g., 8E4.Cells were then washed three times with PBS with 1% BSA, followed byincubation for 1 hour with a 1:500 dilution of the following secondaryantibodies:

-   -   1. Goat Anti-Mouse IgM (μ chain) AlexaFluor568 (Molecular Probes        Cat, No. A21043);    -   2. Goat Anti-Rabbit IgG (H+L) highly cross absorbed        AlexaFluor488 (Molecular Probes Cat. No. Al 1034)        Cells were washed three times in PBS with BSA, mounted, and        stored at 4° C. prior to visualization.

Results

Initial experiments indicated that the concentration ofpolyubiquitinated proteins (PUP) in cells is greater (very intense) inhealthy cells (FIGS. 3A and 3C) than in Gaucher N370S (FIGS. 3D and 3F)and L444P fibroblasts (FIGS. 3G and 3I) where staining is much lessintense). Protein aggregation is known to inhibit theubiquitin/proteasome pathway. Accordingly, decreasing aggregation usingchaperones has a positive effect on the proteasorne-mediated degradationpathway.

Discussion

Gaucher patients with the L444P mutation have extensive CNS involvement.This may be due to the fact that the human L444P mutant enzyme is knownto be much more unstable than, e.g., the N370S mutant, making it evenmore likely that protein aggregates will form, and thereby inhibitingthe ubiquitin/proteasome pathway (Tsuji et al., N. Eng, J. Med. 1987;315: 570). Many other neurodegenerative diseases are caused by mutationswhich result in the accumulation of ubiquitinated proteins, and it hasbeen further reported that protein aggregates may directly impair theubiquitin/proteasome pathway and induce the expression of inflammatorymediators (Li et al., The International Journal of Biochemistry & CellBiology. 2003; 35: 547-552).

If mouse L444P is stabilized using a specific pharmacological chaperone,the stress on the ubiquitin/proteasome pathway is alleviated by theincreased Gba trafficking to the lysosome, thereby elongating thehalf-life of the mutant Gba-instead of being degraded in the ER it wouldtraffick to the lysosome, This explains the increased PUP staining innormal fibroblasts compared to Gaucher fibroblasts.

Other Gba mutations that clinically do not result in overt CNS symptoms(i.e., N370S) may still result in the accumulation of the mutant proteinin the ER and cytosol, causing additional stress on theubiquitin/proteasome pathway or disrupting trafficking in neurons bydecreasing the cells' ability to monoubiquitinate proteins.

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and theaccompanying figures. Such modifications are intended to fall within thescope of the appended claims.

It is further to be understood that all values approximate, and areprovided for description.

Patents, patent applications, publications, product descriptions, andprotocols are cited throughout this application, the disclosures ofwhich are incorporated herein by reference in their entireties for allpurposes.

1-20. (canceled)
 21. A method for treating a neurodegenerative disorderin an individual having or at risk of developing an a-synucleinopathy,wherein the individual has a mutation in the gene encodingβ-glucocerebrosidase, which method comprises administering to theindividual an effective amount of an isofagomine derivative thatreversibly binds to β-glucocerebrosidase.
 22. The method of claim 21,wherein the isofagomine derivative is effective to prevent or reduceneuronal or extraneuronal accumulation of α-synuclein.
 23. The method ofclaim 21, wherein the α-synucleinopathy is Parkinson's or parkinsonism.24. The method of claim 21, wherein the individual is homozygous for themutation.
 25. The method of claim 21, wherein the individual ishemizygous, heterozygous or compound heterozygous for the mutation. 26.The method of claim 25, wherein the individual is heterozygous for an84GG mutation.
 27. The method of claim 25, wherein the individual isheterozygous for an R496H mutation.
 28. The method of claim 21, whereinthe individual is heterozygous or homozygous for an N370S mutation. 29.The method of claim 21, wherein the mutation is a conformational mutant.30. The method of claim 29, wherein the pharmacological chaperoneincreases trafficking of the mutant enzyme from the endoplasmicreticulum and/or restores enzyme activity.
 31. A method for treatingParkinson's or parkinsonism, wherein the individual has a mutation inthe gene encoding β-glucocerebrosidase, which method comprisesadministering to the individual an effective amount of a pharmacologicalchaperone that reversibly binds to β-glucocerebrosidase.
 32. The methodof claim 31, wherein the pharmacological chaperone e is effective toprevent or reduce neuronal or extraneuronal accumulation of α-synuclein.33. The method of claim 31, wherein the pharmacological chaperone isisofagomine or an isofagomine derivative.
 34. The method of claim 31,wherein the pharmacological chaperone is isofagomine.
 35. The method ofclaim 31, wherein the pharmacological chaperone is C-benzyl isofagomine.36. The method of claim 31, wherein the individual is homozygous for themutation.
 37. The method of claim 31, wherein the individual ishemizygous, heterozygous or compound heterozygous for the mutation. 38.The method of claim 37, wherein the individual is heterozygous for an84GG mutation.
 39. The method of claim 37, wherein the individual isheterozygous for an R496H mutation.
 40. The method of claim 31, whereinthe individual is heterozygous or homozygous for an N370S mutation.